Methods, Systems, and Arrays for Biomolecular Analysis

ABSTRACT

Disclosed herein are formulations, substrates, and arrays. Also disclosed herein are methods for manufacturing and using the formulations, substrates, and arrays. Also disclosed are methods for identifying peptide sequences useful for diagnosis and treatment of disorders, and methods for using the peptide sequences for diagnosis and treatment of disorders, e.g., celiac disorder. In certain embodiments, substrates and arrays comprise a porous layer for synthesis and attachment of polymers or biomolecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/707,758 filed Sep. 28, 2012, U.S. Provisional PatentApplication No. 61/732,221, filed Nov. 30, 2012, U.S. Provisional PatentApplication No. 61/805,884, filed Mar. 27, 2013, U.S. Provisional PatentApplication No. 61/765,584, filed Feb. 15, 2013, U.S. Provisional PatentApplication No. 61/866,512, filed Aug. 15, 2013, and InternationalPatent Application No. PCT/US2013/025190, filed Feb. 7, 2013, thedisclosures of which are incorporated by reference in their entirety forall purposes.

BACKGROUND

A typical microarray system is generally comprised of biomolecularprobes, such as DNA, proteins, or peptides, formatted on a solid planarsurface like glass, plastic, or silicon chip, plus the instrumentsneeded to handle samples (automated robotics), to read the reportermolecules (scanners) and analyze the data (bioinformatic tools).Microarray technology can facilitate monitoring of many probes persquare centimeter. Advantages of using multiple probes include, but arenot limited to, speed, adaptability, comprehensiveness and therelatively cheaper cost of high volume manufacturing. The uses of suchan array include, but are not limited to, diagnostic microbiology,including the detection and identification of pathogens, investigationof anti-microbial resistance, epidemiological strain typing,investigation of oncogenes, analysis of microbial infections using hostgenomic expression, and polymorphism profiles.

Recent advances in genomics have culminated in sequencing of entiregenomes of several organisms, including humans. Genomics alone, however,cannot provide a complete understanding of cellular processes that areinvolved in disease, development, and other biological phenomena;because such processes are often directly mediated by polypeptides.Given that huge numbers of polypeptides are encoded by the genome of anorganism, the development of high throughput technologies for analyzingpolypeptides is of paramount importance.

Peptide arrays with distinct analyte-detecting regions or probes can beassembled on a single substrate by techniques well known to one skilledin the art. A variety of methods are available for creating a peptidemicroarray. These methods include: (a) chemo selective immobilizationmethods; and (b) in situ parallel synthesis methods which can be furtherdivided into (1) SPOT synthesis and (2) photolithographic synthesis.

SUMMARY

The invention encompasses, in several embodiments formulations,substrates, and arrays. The invention also includes methods formanufacturing and using the formulations, substrates, and arrays.

In one embodiment, the invention includes a method for obtaining peptidebinding data, comprising: obtaining a peptide array, said arraycomprising at least 10,000 peptide features/square millimeter;contacting said array with a sample comprising a plurality of ligandsfor at least a subset of said 100,000 peptide features under conditionsthat promote ligand binding; and imaging said array to identify bindingof said plurality of ligands to said peptide array.

In some embodiments of the method, a total number of features is atleast about 500,000, 1,000,000, 2,000,000, or 18,000,000. In otherembodiments of the method, said microarray has an area that is less thanor equal to 0.2, 1, 10, 100, or 150 square millimeters. In yet otherembodiments of the method, said sample has a volume that is less than orequal to 100, 50, 10, 5, 1.5, or 1 μL. In some embodiments of themethod, an elapsed time from sample contacting to imaging is less than20, 5, or 1 minutes. In some embodiments of the method, a coefficient ofvariation of data obtained from said array is not greater than 5, 2, or1 percent. In some embodiments of the method, said microarray comprisesat least 1,000,000, 10,000,000, 15,000,000 features per squarecentimeter. In some embodiment of the method, said contacting occurs ata concentration of said plurality of ligands that is within the range ofapproximately 1 pg/ml to approximately 1,000 μg/ml in said sample. Insome embodiment of the method, said imaging comprises identifyingbinding of at least 1,000, 100,000, 1,000,000, 10,000,000, 15,000,000 or100,000,000 ligands to said features of said microarray. In someembodiment of the method, said features are selected from a groupconsisting of proteins, DNA binding sequences, antibodies, peptides,oligonucleotides, nucleic acids, peptide nucleic acids, deoxyribonucleicacids, ribonucleic acids, peptide mimetics, nucleotide mimetics,chelates, and biomarkers.

In one embodiment, the invention includes an inverted pillar plate forassaying microarrays, comprising: a plurality of chip mounts, each chipmount configured to affix at least one of a plurality of microarrays andto prevent the at least one microarray from being displaced from thechip mount when the chip mount is placed facing downwards into a wellcontaining an assay solution; and a plate comprising a plurality ofinverted plate pillars that extend approximately perpendicular from theplate, each inverted plate pillar configured to be coupled to one of theplurality of chip mounts, wherein each chip mount is affixed to at leastone of the plurality of inverted plate pillars so that each chip mountis prevented from being displaced from the at least one inverted platepillar when the plate is turned upside down.

In some embodiment, the invention includes a method of assaying chiparrays, comprising: providing a plurality of chip mounts, each chipmount configured to affix a microarray and to prevent the microarrayfrom being displaced from the chip mount when the chip mount is placedfacing downwards into a well containing an assay solution; affixing aplurality of microarrays onto the chip mounts; providing a pillar platecomprising a plurality of inverted plate pillars that extendapproximately perpendicular from the pillar plate, each plate pillarconfigured to be coupled to one of the plurality of chip mounts;affixing the chip mounts with the affixed microarrays to at least one ofthe plurality of plate pillars so that each chip mount is prevented frombeing displaced from the at least one plate pillar when the pillar plateis turned upside down; and assaying the plurality of microarrays byturning the pillar plate upside down and placing each microarray into awell comprising assay solution.

In some embodiment, the invention includes a method of assuringuniformly high quality of a microarray of features that are attached toa surface of the microarray at positionally-defined locations,comprising: soft-baking the microarray coated with a couplingformulation, the coupling formulation comprising the features;determining the thickness of the soft-baked microarray; responsive tothe thickness of the soft-backed microarray falling outside a firstthreshold range starting over soft-baking the microarray after strippingoff the coat; exposing the soft-baked microarray to light under aphotomask; hard-baking the exposed microarray; and responsive to thethickness of the hard-backed microarray falling outside a secondthreshold range starting over with soft-baking the microarray afterstripping off the coat.

In some embodiment, the invention includes a method of assuringuniformly high quality of a microarray of features that are attached toa surface of the microarray at positionally-defined locations,comprising: soft-baking the microarray coated with a couplingformulation, the coupling formulation comprising the features; exposingthe soft-baked microarray to light under a photomask, the photomaskcomprising a diffusion pattern and a overlay pattern; hard-baking theexposed microarray; and responsive to the diffusion pattern or theoverlay pattern of the hard-backed microarray falling outside atolerance range when compared to a standard diffusion or overlay patternstarting over with soft-baking the microarray after stripping off thecoat.

In one embodiment, the invention includes a method for collecting datafrom a chip array and for piecewise real-time scanning and stitching ofsaid data, comprising steps of: providing a chip array comprising of: aplurality of microarrays, each microarray comprising features that areattached to a surface of the microarray at positionally-definedlocations, aligning a first region of the chip array with a scan mask ofa microscope; imaging the first region of the chip array under the scanmask by the microscope; and rotating, by using a computer processor, theimaged first region of the chip array into standard orientation based onan alignment mark on a surface of a microarray that is at apositionally-defined location within the imaged first region.

In some embodiment, the data-collecting method further comprises stepsof: aligning a second region of the chip array with the scan mask sothat the second region partially overlaps with the first region; imagingthe second region of the chip array under the scan mask by themicroscope; and rotating, by using a computer processor, the imagedsecond region of the chip array into standard orientation based on analignment mark on a surface of a microarray that is at apositionally-defined location within the imaged second region. In someof these embodiments, the method further comprises steps of combiningthe rotated images of the first and second region for analyzing thefeatures located on the surface of the microarrays within the imagedfirst and second region, wherein any overlapping parts of the imagedfirst and second region are averaged for the analysis.

In some embodiment, the data-collecting method further comprises stepsof: storing the rotated images of the first and second region within aimage database.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, embodiments, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1 shows a flow chart for performing an assay using a chip arraywith a robotic device, according to one embodiment.

FIG. 2 shows a flow chart depicting an example of a chip array analysisprocess, according to one embodiment.

FIG. 3 shows a robotic chip array system for performing steps in apacking process of chip arrays, according to one embodiment.

FIG. 4 shows a workbench system for performing steps in an assay usingchip arrays and well plates, according to one embodiment.

FIG. 5 shows a robotic chip array system for performing steps inscanning chip arrays, according to one embodiment.

FIG. 6 shows a flow diagram depicting one embodiment of the diagnosticmodel provided herein, according to one embodiment.

FIG. 7 shows the structure of linker molecules, including e.g.polyethylene glycol (PEG), glycine (GLY) linker chain and a protectinggroup of tert-Butyloxycarbonyl (boc), attached via3-amino-triethoxysilane (APTES) on a single chip for linking a peptideor protein to the surface of the chip to the unprotected NH₂ group,according to one embodiment.

FIG. 8 shows control linker molecules that are the acetylated (CAP)versions of the linker molecules from FIG. 7, according to oneembodiment.

FIG. 9 shows the deprotected linker molecules, i.e. after removing theboc group and leaving the NH₂ group unprotected (not shown), of FIG. 7,according to one embodiment.

FIG. 10 shows a step by step process for adding a protein, e.g. IL-6,and an antibody, e.g. p53 antibody, to a chip, according to oneembodiment.

FIG. 11 shows the binding of protein to linker molecules attached to thesurface of a chip via 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) coupling, according to one embodiment.

FIG. 12 shows the binding of antibody to linker molecules attached tothe surface of a chip via EDC coupling, according to one embodiment.

FIG. 13 shows a process of performing steps in an assay using apolydimethylsiloxane (PDMS) film well plate and each well holding achip, according to one embodiment.

FIG. 14 shows a top view of a PDMS film well plate, according to oneembodiment.

FIG. 15 shows a side view of a PDMS film well plate, according to oneembodiment.

FIG. 16 shows schematics of a chip array with well plates using invertedpillars and its use with a 3×3 well plate in an assay, according to oneembodiment.

FIGS. 17A-E show a chip array structure of multiple chips on top ofpillar caps with the pillar caps attached to a pillar plate, accordingto one embodiment. FIG. 17A shows a chip or a plurality of chips placedonto a pillar cap. FIG. 17B shows the interface of each pillar cap witha pillar plate of 24 pillars. FIG. 17C shows the assembled chip arraystructure. FIGS. 17D and E show the dimensions of a 24-pillar and96-pillar plate, respectively.

FIG. 18 shows results of an assay using a chip comprising IL-6 proteinsimmobilized to the chip surface with different linker molecules placingthe IL-6 proteins at various distances from the chip surface, accordingto one embodiment.

FIG. 19 shows results of an assay using a chip comprising IL-6 proteinsimmobilized to the surface of the chip with different linker molecules,also including acetylated linker molecules as negative controls,according to one embodiment.

FIG. 20 shows results of an assay using a chip comprising p53 antibodiesimmobilized to the chip surface with different linker molecules placingthe p53 antibodies at various distances from the chip surface, accordingto one embodiment.

FIG. 21 shows results of an assay using a chip comprising p53 antibodiesimmobilized to the chip surface with different linker molecules, alsoincluding acetylated linker molecules as negative controls, according toone embodiment.

FIG. 22 shows minimal binding to the acetylated control linkermolecules, according to one embodiment.

FIG. 23 shows an example of an assay using different linker moleculesimmobilizing selected antibodies and/or proteins to the chip surface,according to one embodiment.

FIG. 24 shows a detection range (sensitivity) of a chip over a range ofantibody concentrations, according to one embodiment.

FIG. 25 shows reproducibility of assay results across multiple chips,according to one embodiment.

FIG. 26 shows an ideal and actual layout of chips on a chip array andcorresponding areas on the chip array scanned by confocal microscope,according to one embodiment.

FIG. 27 shows an image of a chip array of three merged channelsincluding reflected light (bright) and two filtered channels (red andgreen), according to one embodiment.

FIG. 28 shows a pixel diagram and an intensity profile of the reflectedlight (bright) channel across a surface of a chip array, according toone embodiment.

FIG. 29 shows a flowchart for calculating an intensity threshold thateliminates background noise on a chip array, according to oneembodiment.

FIG. 30 shows results of using an intensity threshold to identifyregions of interest (ROIs) on a chip array and applying the identifiedROIs to two filter channels (red and green), according to oneembodiment.

FIG. 31 shows the size of a chip's alignment mark in comparison to areason the chip occupied by features, according to one embodiment.

FIG. 32 shows the identification of alignment marks of different chipson a chip array within one scanning area of a confocal microscope,according to one embodiment.

FIG. 33 shows the alignment of a chip array using alignment marks toaccurately identify regions of interest, e.g. a chip, and to positionthe chip for scanning by a confocal microscope (CCD camera), accordingto one embodiment.

FIG. 34 shows a flow chart for position a chip on a chip array bytranslating and rotating the chip based on the actual and the desiredposition of an alignment mark on the chip, according to one embodiment.

FIG. 35 shows the positioning of a chip by theta-angle rotation based onthe position of an alignment mark on the chip, according to oneembodiment.

FIG. 36 shows the first step of mounting the chips in a process flow forcalculating the angle of correction about the center of a chip array tocorrect for misalignment between chips on the chip array, according toone embodiment.

FIG. 37 shows the second step of determining the first rotation angle(θ₁) for a chip in a process flow for calculating the angle ofcorrection about the center of a chip array to correct for misalignmentbetween chips on the chip array, according to one embodiment.

FIG. 38 shows the third step of determining the second rotation angle(θ₂) for a chip in a process flow for calculating the angle ofcorrection about the center of a chip array to correct for misalignmentbetween chips on the chip array, according to one embodiment.

FIG. 39 shows the fourth step of determining rotation angles of anotherchip in a process flow for calculating the angle of correction about thecenter of a chip array to correct for misalignment between chips on thechip array, according to one embodiment.

FIG. 40 shows the stitching of feature data obtained from chips on achip array, according to one embodiment.

FIG. 41 shows flow charts for collecting a chip's signature data, e.g.bar code, alignment marks to determine offsets and rotation angles, foranalyzing chip data from an assay and for stitching together assay datafrom multiple chips on a chip array, according to one embodiment.

FIG. 42 shows a chip array system for performing inline quality controlon a chip array, according to one embodiment.

FIG. 43A-B show the diffusion and overlay test pattern of a photomaskand a standard intensity pattern of a chip under a confocal microscopeafter UV-light exposure with the photomask and baking of the chip,respectively, according to some embodiments.

FIG. 44A-D show standard intensity pattern of a chip as shown in FIG.38B and variations in overlay locations and diffusion amount,respectively, according to some embodiments.

FIGS. 45A-K show intensity profiles for point mutations of peptidesbinding an antibody to determine which amino acids in the peptidesequence are material to binding the antibody, according to someembodiments. The original peptide sequence of amino acids is shown insingle letter code along the top of each intensity profile with thecorresponding amino acid replacement (point mutation) along the verticalaxis. FIG. 41A includes a chart that translates measured intensities toa grey-scale displayed in the intensity profiles.

FIG. 46 shows end-of-line BioQC, according to one embodiment.

FIG. 47 shows end-of-line BioQC for Citrulline, according to oneembodiment.

DETAILED DESCRIPTION Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

As used herein the term “wafer” refers to a slice of semiconductormaterial, such as a silicon or a germanium crystal generally used in thefabrication of integrated circuits. Wafers can be in a variety of sizesfrom, e.g., 25.4 mm (1 inch) to 300 mm (11.8 inches) along one dimensionwith thickness from, e.g., 275 μm to 775 μm.

As used herein the term “photoresist” or “resist” or “photoactivematerial” refers to a light-sensitive material that changes itssolubility in a solution when exposed to ultra violet or deep ultraviolet radiation. Photoresists are organic or inorganic compounds thatare typically divided into two types: positive resists and negativeresists. A positive resist is a type of photoresist in which the portionof the photoresist that is exposed to light becomes soluble to thephotoresist developer. The portion of the photoresist that is unexposedremains insoluble to the photoresist developer. A negative resist is atype of photoresist in which the portion of the photoresist that isexposed to light becomes insoluble to the photoresist developer. Theunexposed portion of the photoresist is dissolved by the photoresistdeveloper.

As used herein the term “photomask” or “reticle” or “mask” refers to anopaque plate with transparent patterns or holes that allow light to passthrough. In a typical exposing process, the pattern on a photomask istransferred onto a photoresist.

As used herein the term “coupling molecule” includes in one embodimentany natural or artificially synthesized amino acid with its amino groupprotected with a fluorenylmethyloxycarbonyl (Fmoc) ortert-Butyloxycarbonyl (boc) group. These amino acids may optionally havetheir side chains protected. Examples of coupling molecules include, butare not limited to, boc-Gly-COOH, Fmoc-Trp-COOH. Other embodiments ofcoupling molecules include monomer molecules and combinations thereofthat can form polymers upon coupling, e.g., nucleotides, sugars and thelike, and are described below.

As used here in the term “coupling” or “coupling process” or “couplingstep” refers to a process of forming a bond between two or moremolecules such as a linker molecule or a coupling molecule. A bond canbe a covalent bond such as a peptide bond. A peptide bond can a chemicalbond formed between two molecules when the carboxyl group of onecoupling molecule reacts with the amino group of the other couplingmolecule, releasing a molecule of water (H₂O). This is a dehydrationsynthesis reaction (also known as a condensation reaction), and usuallyoccurs between amino acids. The resulting —C(═O)NH— bond is called apeptide bond, and the resulting molecule is an amide.

As used herein the terms “polypeptide,” “peptide,” or “protein” are usedinterchangeably to describe a chain or polymer of amino acids that arelinked together by bonds. Accordingly, the term “peptide” as used hereinincludes a dipeptide, tripeptide, oligopeptide, and polypeptide. Theterm “peptide” is not limited to any particular number of amino acids.In some embodiments, a peptide contains about 2 to about 50 amino acids,about 5 to about 40 amino acids, or about 5 to about 20 amino acids. Amolecule, such as a protein or polypeptide, including an enzyme, can bea “native” or “wild-type” molecule, meaning that it occurs naturally innature; or it may be a “mutant,” “variant,” “derivative,” or“modification,” meaning that it has been made, altered, derived, or isin some way different or changed from a native molecule or from anothermolecule such as a mutant. A “point mutation” refers to the mutation ofone amino acid among the amino acids in a sequence of a peptide.

As used herein the term “biomarkers” includes, but is not limited toDNA, RNA, proteins (e.g., enzymes such as kinases), peptides, sugars,salts, fats, lipids, ions and the like.

As used herein the term “linker molecule” or “spacer molecule” includesany molecule that does not add any functionality to the resultingpeptide but spaces and extends out the peptide from the substrate, thusincreasing the distance between the substrate surface and the growingpeptide. This generally reduces steric hindrance with the substrate forreactions involving the peptide (including uni-molecular foldingreactions and multi-molecular binding reactions) and so improvesperformance of assays measuring one or more embodiments of peptidefunctionality.

As used herein the term “developer” refers to a solution that canselectively dissolve the materials that are either exposed or notexposed to light. Typically developers are water-based solutions withminute quantities of a base added. Examples include tetramethyl ammoniumhydroxide in water-based developers. Developers are used for the initialpattern definition where a commercial photoresist is used. Use ofdevelopers is described in Example 1 below.

As used herein the term “protecting group” includes a group that isintroduced into a molecule by chemical modification of a functionalgroup in order to obtain chemoselectivity in a subsequent chemicalreaction. “Chemoselectivity” refers to directing a chemical reactionalong a desired path to obtain a pre-selected product as compared toanother. For example, the use of boc as a protecting group enableschemoselectivity for peptide synthesis using a light mask and aphotoacid generator to selectively remove the protecting group anddirect pre-determined peptide coupling reactions to occur at locationsdefined by the light mask.

As used herein the term “microarray,” “array” or “chip” refers to asubstrate on which a plurality of probe molecules of protein or specificDNA binding sequences have been affixed at separate locations in anordered manner thus forming a microscopic array. Protein or specific DNAbinding sequences may be bound to the substrate of the chip through oneor more different types of linker molecules. A “chip array” refers to aplate having a plurality of chips, for example, 24, 96, or 384 chips.

As used herein the term “probe molecules” refers to, but is not limitedto, proteins, DNA binding sequences, antibodies, peptides,oligonucleotides, nucleic acids, peptide nucleic acids (“PNA”),deoxyribonucleic acids (DNA), ribonucleic acids (RNA), peptide mimetics,nucleotide mimetics, chelates, biomarkers and the like. As used herein,the term “feature” refers to a particular probe molecule that has beenattached to a microarray. As used herein, the term “ligand” refers to amolecule, agent, analyte or compound of interest that can bind to one ormore features.

As used herein the term “microarray system” or a “chip array system”refers to a system usually comprised of probe molecules formatted on asolid planar surface like glass, plastic or silicon chip plus theinstruments needed to handle samples (automated robotics), to read thereporter molecules (scanners) and analyze the data (bioinformatictools).

As used herein the term “patterned region” or “pattern” or “location”refers to a region on the substrate on which are grown differentfeatures. These patterns can be defined using photomasks.

As used herein the term “derivatization” refers to the process ofchemically modifying a surface to make it suitable for bio molecularsynthesis. Typically derivatization includes the following steps: makingthe substrate hydrophilic, adding an amino silane group, and attaching alinker molecule.

As used herein the term “capping” or “capping process” or “capping step”refers to the addition of a molecule that prevents the further reactionof the molecule to which it is attached. For example, to prevent thefurther formation of a peptide bond, the amino groups are typicallycapped by acetylation in the presence of an acetic anhydride molecule.

As used herein the term “diffusion” refers to the spread of, e.g.,photoacid through random motion from regions of higher concentration toregions of lower concentration.

As used herein the term “dye molecule” refers to a dye which typicallyis a colored substance that can bind to a substrate. Dye molecules canbe useful in detecting binding between a feature on an array and aligand, e.g. a molecule of interest.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific.

As used herein the term “biological sample” refers to a sample derivedfrom biological tissue or fluid that can be assayed for an analyte(s) ofinterest or any ligand. Such samples include, but are not limited to,sputum, amniotic fluid, blood, blood cells (e.g., white cells), tissueor fine needle biopsy samples, urine, peritoneal fluid, and pleuralfluid, or cells therefrom. Biological samples may also include sectionsof tissues such as frozen sections taken for histological purposes.Although the sample is typically taken from a human patient, the assayscan be used to detect analyte(s) of interest in samples from anyorganism (e.g., mammal, bacteria, virus, algae, or yeast) or mammal,such as dogs, cats, sheep, cattle, and pigs. The sample may bepretreated as necessary by dilution in an appropriate buffer solution orconcentrated, if desired.

As used herein, the term “assay” refers to a type of biochemical testthat measures the presence or concentration of a substance of interestin solutions that can contain a complex mixture of substances.

The term “antigen” as used herein refers to a molecule that triggers animmune response by the immune system of a subject, e.g., the productionof an antibody by the immune system. Antigens can be exogenous,endogenous or auto antigens. Exogenous antigens are those that haveentered the body from outside through inhalation, ingestion orinjection. Endogenous antigens are those that have been generated withinpreviously-normal cells as a result of normal cell metabolism, orbecause of viral or intracellular bacterial infection. Auto antigens arethose that are normal protein or protein complex present in the hostbody but can stimulate an immune response.

As used herein the term “epitope” or “immunoactive regions” refers todistinct molecular surface features of an antigen capable of being boundby component of the adaptive immune system, e.g., an antibody or T cellreceptor. Antigenic molecules can present several surface features thatcan act as points of interaction for specific antibodies. Any suchdistinct molecular feature can constitute an epitope. Therefore,antigens have the potential to be bound by several distinct antibodies,each of which is specific to a particular epitope.

As used herein the term “antibody” or “immunoglobulin molecule” refersto a molecule naturally secreted by a particular type of cells of theimmune system: B cells. There are five different, naturally occurringisotypes of antibodies, namely: IgA, IgM, IgG, IgD, and IgE.

The term percent “identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, thepercent “identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Compositions

Substrates

Also disclosed herein are substrates. In some embodiments, a substratecomprises a planar (e.g., 2-dimensional) layer. In some embodiments, thesurface of a substrate comprises pillars for attachment or synthesis ofmolecules, e.g. peptides, or a first monomer building block. In otherembodiments, a substrate includes a porous (i.e., a 3-dimensional) layercomprising functional groups for binding a first monomer building block.In some embodiments, a porous layer is added to the top of the pillars.In some embodiments, the substrate comprises a porous layer coupled tothe planar layer. In other embodiments, the substrate comprises aplurality of pillars coupled to the planar layer.

In some embodiment, the planar layer can comprise any metal or plasticor silicon or silicon oxide or silicon nitride. In some embodiment, theplanar layer has an upper surface and a lower surface. In someembodiments, the support layer is 1,000-2,000 angstroms thick. In someembodiments, the planar layer is about less than 500, 1,000, 2,000,3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000,or greater than 12,000 angstroms thick (or any integer in between). Insome embodiments, the metal is chromium. In some embodiments, the metalis chromium, titanium, aluminum, tungsten, gold, silver, tin, lead,thallium, indium, or a combination thereof. In some embodiments, theplanar layer is at least 98.5-99% metal. In some embodiments, the planarlayer is 100% metal. In some embodiments, the planar layer is at leastabout greater than 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, or 99%metal. In some embodiments, the planar layer is a homogenous layer ofmetal.

In some embodiments, a substrate surface is derivatized with freecarboxylic acid groups. In other embodiments, a substrate surface isderivatized with free amine groups. In yet other embodiments, asubstrate surface is derivatized with other free functional groups forsolid state synthesis. A surface that is derivatized with free aminegroups can be converted to free carboxylic acid groups by reacting theamine with one carboxylic acid group of a molecule having at least twofree carboxylic acid groups. For example, by using carbodiimide onecarboxylic acid group is first activated to form an intermediateO-acylisourea that then further reacts with the free amine groups for anamide bond and attached to the substrate surface. In some embodiments,the molecule with multiple carboxylic acid groups includes, but is notlimited to, succinic anhydride, polyethylene glycol diacid,benzene-1,3,5-tricarboxylic acid, benzenehexacarboxylic acid andcarboxymethyl dextran. For example, the free carboxylic acid or freeamine groups bind amino acids, peptides or proteins during peptidesynthesis and protein coupling. In another example, the free functionalgroups bind to linker molecules that couple (“link”) other probemolecules or biomarkers to the substrate. In some embodiments, acoupling molecule is attached to the surface of at least one pillar. Inother embodiments, a coupling molecule is attached to the surface ofeach pillar.

In some embodiments, a polymer is in contact with the surface of atleast one of said pillars. In other embodiments, a polymer is in contactwith the surface of each pillar. In some embodiments, a gelatinous formof a polymer is in contact with the surface of at least one of saidpillars. In some embodiments, a solid form of a water soluble polymer isin contact with the surface of at least one of said pillars.

In some embodiments, the substrate surface comprises silicon dioxide forcontacting the surface with a photoactive coupling formulationcomprising a photoactive compound, a coupling molecule, a couplingreagent, a polymer, and a solvent, wherein the contracting is followedby applying ultraviolet light to positionally-defined locations locatedon the top of the surface and in contact with the photoactive couplingformulation.

In some embodiments, the substrate surface is a material or group ofmaterials having rigidity or semi-rigidity. In some embodiments, thesubstrate surface can be substantially flat, although in someembodiments it can be desirable to physically separate synthesis regionsfor different molecules or features with, for example, wells, raisedregions, pins, pillars, etched trenches, or the like. In certainembodiments, the substrate surface may be porous. Surface materials caninclude, for example, silicon, bio-compatible polymers such as, forexample poly(methyl-methacrylate) (PMMA) and polydimethylsiloxane(PDMS), glass, SiO₂ (such as a thermal oxide silicon wafer used by thesemiconductor industry), quartz, silicon nitride, functionalized glass,gold, platinum, and aluminum.

Derivatized substrate surfaces include, for example, amino-derivatizedglass, carboxy-derivatized glass, and hydroxyl-derivatized glass.Additionally, a surface may optionally be coated with one or more layersto provide a second surface for molecular attachment or derivatization,increased or decreased reactivity, binding detection, or otherspecialized application. Substrate surface materials and/or layer(s) canbe porous or non-porous. For example, a substrate surface comprisesporous silicon.

Pillar Substrate

In some embodiments, a substrate comprises a planar layer comprising ametal and having an upper surface and a lower surface; and a pluralityof pillars operatively coupled to the planar layer inpositionally-defined locations, wherein each pillar has a planar surfaceextended from the planar layer, wherein the distance between the surfaceof each pillar and the upper surface of the planar layer is betweenabout 1,000-5,000 angstroms, and wherein the plurality of pillars arepresent at a density of greater than about 10,000/cm². In otherembodiments, the distance between the surface of each pillar and theupper surface of the planar layer can be between about less than 1,000,2,000, 3,000, 3,500, 4,500, 5,000, or greater than 5,000 angstroms (orany integer in between).

In some embodiments, the surface of each pillar is parallel to the uppersurface of the planar layer. In some embodiments, the surface of eachpillar is substantially parallel to the upper surface of the planarlayer.

In some embodiments, the distance between the surface of each pillar andthe lower surface of the planar layer is 2,000-7,000 angstroms. In otherembodiments, the distance between the surface of each pillar and thelower surface of the planar layer is about less than 500, 1,000, 2,000,3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000,or greater than 12,000 angstroms (or any integer in between). In yetother embodiments, the distance between the surface of each pillar andthe lower surface of the planar layer is 7,000, 3,000, 4,000, 5,000,6,000, or 7,000 angstroms (or any integer in between).

In some embodiments, the plurality of pillars are present at a densityof greater than 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000,8,000, 9,000, 10,000, 11,000, or 12,000/cm² (or any integer in between).In other embodiments, the plurality of pillars are present at a densityof greater than 10,000/cm². In yet other embodiments, the plurality ofpillars are present at a density of about 10,000/cm² to about 2.5million/cm² (or any integer in between). In some embodiments, theplurality of pillars are present at a density of greater than 2.5million/cm².

In some embodiments, the surface area of each pillar surface is at least1 μm². In other embodiments, the surface area of each pillar surface canbe at least 0.1, 0.5, 12, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, or 50 μm² (or any integer in between). In yet other embodiments,the surface area of each pillar surface has a total area of less than10,000 μm².

In yet other embodiments, the surface area of each pillar surface has atotal area of less than 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 11,000, or 12,000 μm² (or any integer inbetween). In some embodiments, the surface of each pillar is square orrectangular in shape.

In some embodiments, the center of each pillar is at least 2,000angstroms from the center of any other pillar. In other embodiments, thecenter of each pillar is at least about 500, 1,000, 2,000, 3,000, or4,000 angstroms (or any integer in between) from the center of any otherpillar. In yet other embodiments, the center of each pillar is at leastabout 2 μm to 200 μm from the center of any other pillar.

In some embodiments, at least one or each pillar comprises silicon. Inother embodiments, at least one or each pillar comprises silicon dioxideor silicon nitride. In some of these embodiments, at least one or eachpillar is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 98.5, or 99%silicon dioxide.

In some embodiments, the metal of the planar layer is chromium. In otherembodiments, the metal is chromium, titanium, aluminum, tungsten, gold,silver, tin, lead, thallium, indium, or a combination thereof. In someembodiments, the planar layer is at least 98.5-99% (by weight) metal. Inother embodiments, the planar layer is 100% metal. In yet otherembodiments, the planar layer is at least about greater than 90, 91, 92,93, 94, 95, 96, 97, 98, 98.5, or 99% metal. In some embodiments, theplanar layer is a homogenous layer of metal.

In some embodiments, the surface of at least one of said pillars of thesubstrate is derivatized. In some embodiments, a substrate can include apolymer chain attached to the surface of at least one of said pillars.In some embodiments, the polymer chain comprises a peptide chain. Insome embodiments, the attachment to the surface of said at least onepillar is via a covalent bond.

In some embodiments, the substrate can be coupled to a silicon dioxidelayer. The silicon dioxide layer can be about 0.5 μm to 3 μm thick. Insome embodiments, the substrate can be coupled to a wafer, e.g., asilicon wafer. The silicon wafer can be about 700 μm to 750 μm thick.

Porous Layers Substrate

In another embodiments, a substrate comprises a porous layer coupled toa plurality of pillars, wherein the porous layer comprises functionalgroups for attachment of a molecule to the substrate, and wherein theplurality of pillars are coupled to a planar layer inpositionally-defined locations, each pillar having a planar surfaceextended from the planar layer by the distance between the surface ofeach pillar and the upper surface of the planar layer that is betweenabout 1,000-5,000 angstroms, and the plurality of pillars are present ata density of greater than about 10,000/cm².

Porous layers that can be used are flat, permeable, polymeric materialsof porous structure that have a carboxylic acid functional group (thatis native to the constituent polymer or that is introduced to the porouslayer) for attachment of the first peptide building block. For example,a porous layer can be comprised of porous silicon with functional groupsfor attachment of a polymer building block attached to the surface ofthe porous silicon. In another example, a porous layer can comprise across-linked polymeric material. In some embodiments, the porous layercan employ polystyrenes, saccharose, dextrans, polyacryloylmorpholine,polyacrylates, polymethylacrylates, polyacrylamides,polyacrylolpyrrolidone, polyvinylacetates, polyethyleneglycol, agaroses,sepharose, other conventional chromatography type materials andderivatives and mixtures thereof. In some embodiments, the porous layerbuilding material is selected from: poly(vinyl alcohol), dextran, sodiumalginate, poly(aspartic acid), poly(ethylene glycol), poly(ethyleneoxide), poly(vinyl pyrrolidone), poly(acrylic acid), poly(acrylicacid)-sodium salt, poly(acrylamide), poly(N-isopropyl acrylamide),poly(hydroxyethyl acrylate), poly(acrylic acid), poly(sodium styrenesulfonate), poly(2-acrylamido-2-methyl-1-propanesulfonic acid),polysaccharides, and cellulose derivatives. Preferably the porous layerhas a porosity of 10-80%. In one embodiment, the thickness of the porouslayer ranges from 0.01 μm to about 1,000 μm. Pore sizes included in theporous layer may range from 2 nm to about 100 μm.

In another embodiment the porous layer comprises a porous polymericmaterial having a porosity from 10-80%, wherein reactive groups arechemically bound to the pore surfaces and are adapted in use tointeract, e.g. by binding chemically, with a reactive species, e.g.,deprotected monomeric building blocks or polymeric chains. In oneembodiment the reactive group is a free carboxylic acid or a free aminegroup. For example, the carboxylic acid group is free to bind anunprotected amine group of an amino acid, peptide or polypeptide forpeptide synthesis.

Linker Molecules

In some embodiments, the substrate surface is coupled to a plurality oflinker molecules. A linker molecule is a molecule inserted between asubstrate surface disclosed herein and a first coupling molecule that ise.g. the N-terminal amino acid of a peptide being synthesized. A linkermolecule does not necessarily convey functionality to the resultingpeptide, such as molecular recognition functionality, but can insteadelongate the distance between the surface and the synthesized peptide toenhance the exposure of the peptide's functionality region(s) on thesurface.

In some embodiments, a linker can be about 4 to about 40 atoms long toprovide exposure. The linker molecules can be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units,diamines, diacids, amino acids, and combinations thereof. Examples ofdiamines include ethylene diamine and diamino propane. Alternatively,linkers can be the same molecule type as that being synthesized (e.g.,nascent polymers or various coupling molecules), such as polypeptidesand polymers of amino acid derivatives such as for example, aminohexanoic acids. In some embodiments, a linker molecule is a moleculehaving a carboxylic group at a first end of the molecule and aprotecting group at a second end of the molecule. In some embodiments,the protecting group is a boc or Fmoc protecting group. In someembodiments, a linker molecule comprises an aryl-acetylene, apolyethyleneglycol (PEGs), a nascent polypeptide, a diamine, a diacid, apeptide, or combinations thereof.

The unbound portion of a linker molecule, or free end of the linkermolecule, can have a reactive functional group which is blocked,protected, or otherwise made unavailable for reaction by a removableprotective group, e.g., boc or Fmoc as noted above. The protecting groupcan be bound to a monomer, a polymer, or a linker molecule to protect areactive functionality on the monomer, polymer, or linker molecule.Protective groups that can be used include all acid and base labileprotecting groups. For example, peptide amine groups can be protected bytert-butyloxycarbonyl (boc) or benzyloxycarbonyl (CBZ), both of whichare acid labile, or by 9-fluorenylmethoxycarbonyl (Fmoc), which is baselabile.

Additional protecting groups that can be used include acid labile groupsfor protecting amino moieties: tert-amyloxycarbonyl,adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl,2-(p-biphenyl)propyl(2)oxycarbonyl,2-(p-phenylazophenylyl)propyl(2)oxycarbonyl,alpha,alpha-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl,2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl,furfuryloxycarbonyl, triphenylmethyl (trityl),p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl,diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl,and 1-naphthylidene; as base labile groups for protecting aminomoieties: 9 fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl,and 5-benzisoazolylmethyleneoxycarbonyl; as groups for protecting aminomoieties that are labile when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; as groups for protecting aminomoieties that are labile when oxidized: (ethylthio)carbonyl; as groupsfor protecting amino moieties that are labile to miscellaneous reagents,the appropriate agent is listed in parenthesis after the group:phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl(2-aminothiophenol); acid labile groups for protecting carboxylic acids:tert-butyl ester; acid labile groups for protecting hydroxyl groups:dimethyltrityl. (See also, Greene, T. W., Protective Groups in OrganicSynthesis, Wiley-Interscience, NY, (1981)).

In some embodiments, the linker molecule is silane-(boc), where (boc)represents a tert-butyloxycarbonyl protecting group. In someembodiments, the linker molecule is silane-Gly-PEG(boc). In someembodiments, the linker molecule is silane-Gly-PEG-PEG(boc). In someembodiments, the linker molecule is silane-Gly-(PEG(boc))₂. In someembodiments, the linker molecule is silane-PEG-Gly(boc). In someembodiments, the linker molecule is silane-Gly-cyc-PEG(boc), whereGly-cyc represents a glycine chain with a cyclic glycine chainconformation. In some embodiments, the linker molecule issilane-Gly-(PEG(boc))₄.

In some embodiments, linker molecules attached to the surface of eachpillar of the pillar substrate described above comprise a free amine orfree carboxylic acid group. In other embodiments, linker moleculesattached to the surface of at least one pillar of the pillar substratecomprise a free amine or free carboxylic acid group. In someembodiments, a linker molecule having a protecting group is attached tothe surface of each pillar. In other embodiments, a linker moleculehaving a protecting group is attached to the surface of at least onepillar.

Linker Formulations

Also disclosed herein is a linker formulation used for reacting a linkermolecule with the substrate. A linker formulation can include componentssuch as a linker molecule, a polymer, a solvent and a coupling reagent.

In some embodiments, a linker molecule is about 0.5-5 weight % of thetotal formulation concentration. In some embodiments, a linker moleculeis about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, orgreater than 5.0 weight % of the total formulation concentration.

In some embodiments, the polymer is 1 weight % polyvinyl alcohol and 2.5weight % poly vinyl pyrrollidone, the linker molecule is 1.25 weight %polyethylene oxide, the coupling reagent is 1 weight %1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and the solvent includeswater. In some embodiments, the polymer is 0.5-5 weight % polyvinylalcohol and 0.5-5 weight % poly vinyl pyrrollidone, the linker moleculeis 0.5-5 weight % polyethylene oxide, the coupling reagent is 0.5-5weight % 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solventincludes water.

In some embodiments, the polymer is a polyvinyl pyrrolidone and/or apolyvinyl alcohol. The general structure of polyvinyl alcohol is asfollows, where n is any positive integer greater than 1:

In some embodiments, the polymer is about 0.5-5 weight % of the totalformulation concentration. In some embodiments, a water soluble polymeris about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, orgreater than 5.0 weight % of the total formulation concentration.

In some embodiments, the solvent is water, an organic solvent, or acombination thereof. In some embodiments, the organic solvent isN-methyl pyrrolidone, dimethyl formamide, dichloromethane, dimethylsulfoxide, or a combination thereof. In some embodiments, the solvent isabout 80-90 weight % of the total formulation concentration. In someembodiments, the solvent is about less than 70, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or greater than 99 weight % of the totalformulation concentration.

In some embodiments, the coupling reagent is carbodiimide. In someembodiments, a coupling reagent is a water soluble triazole. In someembodiments, a coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the coupling reagent is about 0.5-5weight % of the total formulation concentration. In some embodiments,the coupling reagent is about less than 0.1, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, or greater than 5.0 weight % of the total formulationconcentration.

Microarrays

Also disclosed herein are microarrays. Embodiments of a microarray(“chip”) comprise a substrate and features attached to the substratesurface at positionally-defined locations.

In some embodiments, a microarray comprises two-dimensional array,wherein the positionally-defined locations occupy a 2-dimensional plane.For example, each feature can comprise: a collection of peptide chainsof determinable sequence and intended length, wherein within anindividual feature, the fraction of peptide chains within saidcollection having the intended length is characterized by an averagecoupling efficiency for each coupling step of about 98%. In someembodiments, the average coupling efficiency for each coupling step isat least 98.5%. In some embodiments, the average coupling efficiency foreach coupling step is at least 99%. In some embodiments, the averagecoupling efficiency for each coupling step is at least 90, 91, 92, 93,94, 95, 96, 97, 98, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2,99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100%.

In some embodiments, the features attached to the substrate surface areselected from a group consisting of: proteins, DNA binding sequences,antibodies, peptides, oligonucleotides, nucleic acids, peptide nucleicacids, deoxyribonucleic acids, ribonucleic acids, peptide mimetics,nucleotide mimetics, chelates, biomarkers, and the like.

In some embodiments, the substrate surface of the microarray isfunctionalized with free amine or free carboxylic acids for polypeptidesynthesis. In some embodiments, the free carboxylic acids are activatedto bind to amine groups, e.g., during polypeptide synthesis on thesurface of the microarray.

In some embodiments, the surface density of features on the microarrayis greater than 10/cm², 100/cm², 1,000/cm², 10,000/cm², 100,000/cm²,1,000,000/cm², 10,000,000/cm² or 20,000,000/cm². In some embodiments,the total number of features on the microarray is at least about100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000,900,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000,6,000,000, 7,000,000, 8,000,000, 10,000,000, 12,000,000, 14,000,000,16,000,000, or 18,000,000. In other embodiments, the size of themicroarray is less than or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1,000square millimeters.

In some embodiments, a microarray can be a three-dimensional array,e.g., the substrate comprising a porous layer with features attached tothe surface of the porous layer. In some embodiments, the surface of aporous layer includes external surfaces and surfaces defining porevolume within the porous layer. In some embodiments, a three-dimensionalmicroarray can include features attached to a surface atpositionally-defined locations, said features each comprising: acollection of peptide chains of determinable sequence and intendedlength. In one embodiment, within an individual feature, the fraction ofpeptide chains within said collection having the intended length ischaracterized by an average coupling efficiency for each coupling stepof greater than 98%. In some embodiments, the average couplingefficiency for each coupling step is at least 90, 91, 92, 93, 94, 95,96, 97, 98, 98.5, 98.6, 98.7, 98.8, 98.9, 99.0, 99.1, 99.2, 99.3, 99.4,99.5, 99.6, 99.7, 99.8, 99.9, or 100%.

In some embodiments, each peptide chain is from 5 to 60 amino acids inlength. In some embodiments, each peptide chain is at least 5 aminoacids in length. In some embodiments, each peptide chain is at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids in length. Insome embodiments, each peptide chain is less than 5, at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, orgreater than 60 amino acids in length. In some embodiments, each peptidechain comprises one or more L amino acids. In some embodiments, eachpeptide chain comprises one or more D amino acids. In some embodiments,each peptide chain comprises one or more naturally occurring aminoacids. In some embodiments, each peptide chain comprises one or moresynthetic amino acids.

In some embodiments, a microarray can include at least 1,000 differentpeptide chains attached to the surface. In some embodiments, amicroarray can include at least 10,000 different peptide chains attachedto the surface. In some embodiments, a microarray can include at least100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000,or greater than 10,000 different peptide chains attached to the surface(or any integer in between).

In some embodiments, a microarray can include a single protein, peptidechain, or antibody attached to a plurality of different types of linkermolecules. In some embodiments a microarray can include at least 2different types of linker molecules. In some embodiments, a microarraycan include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50,75, or greater than 100 different types of linker molecules attached tothe substrate.

In some embodiments, each of the positionally-defined locations is at adifferent, known location that is physically separated from each of theother positionally-defined locations. In some embodiments, each of thepositionally-defined locations is a positionally-distinguishablelocation. In some embodiments, each determinable sequence is a knownsequence. In some embodiments, each determinable sequence is a distinctsequence.

In some embodiments, the features are covalently attached to thesurface. In some embodiments, said peptide chains are attached to thesurface through a linker molecule or a coupling molecule.

In some embodiments, the features comprise a plurality of distinct,nested, overlapping peptide chains comprising subsequences derived froma source protein having a known sequence. In some embodiments, eachpeptide chain in the plurality is substantially the same length. In someembodiments, each peptide chain in the plurality is the same length. Insome embodiments, each peptide chain in the plurality is at least 5amino acids in length. In some embodiments, each peptide chain in theplurality is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60amino acids in length. In some embodiments, each peptide chain in theplurality is less than 5, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or greater than 60 amino acidsin length.

In some embodiments, at least one peptide chain in the plurality is atleast 5 amino acids in length. In some embodiments, at least one peptidechain in the plurality is at least 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, or 60 amino acids in length. In some embodiments, at least onepeptide chain in the plurality is less than 5, at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or greaterthan 60 amino acids in length. In some embodiments, each polypeptide ina feature is substantially the same length. In some embodiments, eachpolypeptide in a feature is the same length. In some embodiments, thefeatures comprise a plurality of peptide chains each having a random,determinable sequence of amino acids.

Chip Arrays

Also disclosed herein are chip arrays. In some embodiments, a chip arrayis a two-dimensional array of microarrays (“chips”) on a support layeror plate. In some embodiments of chip arrays, each chip only comprises asingle protein or antibody. In other embodiments, each chip comprises aplurality of proteins, antibodies, peptides, oligonucleotides, DNA, RNA,peptide nucleic acid (“PNA”), probe molecules and the like. In someembodiments, chips are packaged onto a 96 well plate. In someembodiments, epoxy is used to attach a chip to the waiver. In someembodiments, the support layer is an array of pillars, and a chip or aplurality of chips is attached to each pillar. These pillars of thesupport layer are of macroscopic scale and are to be distinguished fromthe substrate pillars described above. In other embodiments, a chip isattached to a cap which attaches to a pillar on a pillar plate.

In one embodiment, chips are formed on a silicon wafer, the siliconwafer being the support layer, and then diced into multiple chips ofvarying dimensions (FIG. 1). In some embodiments, each chip has adimension of 1 mm by 1 mm up to 2 cm to 2 cm. In some embodiments, thechips formed on a wafer and diced into multiple chips fit onto 24-, 96-,192-, or 384-well plates, or any other custom made plates. In someembodiments, these plates have a plurality of wells which act ascontainers for each chip. In some embodiments, the plate is used forin-vitro diagnostics, such as protein-protein interaction assays orother enzymatic reactions.

Robotic Chip Array System

Shown in FIG. 1 is a flow chart for performing an assay using a chiparray with a robotic system. In some embodiments, the assay station isautomated to perform liquid handling on the chip array. In someembodiments, the liquid handling assay station is any commerciallyavailable one that can use the standard or custom made well plates whichhold the plurality of chips. After performing the assay using a liquidhandling assay station, the chip is scanned using any commerciallyavailable confocal or CCD scanner. In some embodiments, the confocalscanner scans multiple chips loaded onto the substrate. In someembodiments, the data from the confocal scanner is analyzed on a VibrantBio Analyzer.

In some embodiments, one or several autoloader units feed the plate tothe liquid handling assay station. Once the assay is performed, thechips are scanned on the confocal scanner using an autoloader. In someembodiments, one or several confocal scanners are connected to theautoloader to allow the autoloader to transfer chip arrays to a one or aplurality of scanners. A flow chart depicting an example of the chiparray analysis process is shown in FIG. 2.

FIG. 3 illustrates the packaging process of the chips including thesteps of: dicing the quality controlled processed wafer into chips,picking the diced chips from the diced wafer and placing them onto atape, picking the chips from the tape and attaching them onto a pillarplate using adhesive, and storing the pillar plates with chips attachedto each pillar for future use.

FIG. 4 illustrates the bioassay process of the pillar plates with chipsattached to each plate including the steps of: placing and washing thepillar plate in a first well plate filled methanol, picking up thepillar plate from the first well plate and transporting it to a secondwell plate filled with TBS for washing. In the third step, the processplaces the pillar plate in third well plate for incubation with theprimary antibody, followed by washing the pillar plate in a fourth wellplate containing PBST. The next step includes placing the pillar platein a fifth well plate for incubation with the secondary antibody,followed by washing the pillar plate in a sixth well plate with PBST andthen by washing it in a seventh well plate with DI water before dryingthe pillar plate in nitrogen for further analysis.

FIG. 5 illustrates the scanning process of the assayed chips includingthe steps of: checking the chips under the microscope to determine ifthey are clean and ready for scanning, washing the chips in DI water ifthe chips are determined to be contaminated, scanning the chips by usinga confocal scanner microscope to determine the signal intensity for eachfeature located on the chips.

Methods

Method of Manufacturing Substrates

Also disclosed herein are methods for making substrates. In someembodiments, a method of producing a substrate can include coupling aporous layer to a support layer. The support layer can comprise anymetal or plastic or silicon or silicon oxide or silicon nitride. In oneembodiment, the substrate comprises multiple carboxylic acid substratesattached to the substrate for binding peptides during peptide synthesisand protein coupling. In some embodiments, a method of producing asubstrate can include coupling a porous layer to a plurality ofsubstrate pillars, wherein the porous layer comprises functional groupsfor attachment of a compound to the substrate, wherein the plurality ofsubstrate pillars are coupled to a planar layer in positionally-definedlocations, wherein each substrate pillar has a planar surface extendedfrom the planar layer, wherein the distance between the surface of eachsubstrate pillar and the upper surface of the planar layer is betweenabout 1,000-5,000 angstroms, and wherein the plurality of substratepillars are present at a density of greater than about 10,000/cm².

In some embodiments, the surface of each substrate pillar is parallel tothe upper surface of the planar layer. In some embodiments, the surfaceof each substrate pillar is substantially parallel to the upper surfaceof the planar layer.

In some embodiments, a method of preparing a substrate surface caninclude obtaining a surface comprising silicon dioxide and contactingthe surface with a photoactive coupling formulation comprising aphotoactive compound, a coupling molecule, a coupling reagent, apolymer, and a solvent; and applying ultraviolet light topositionally-defined locations located on the top of the surface and incontact with the photoactive formulation.

Methods of Manufacturing Microarrays

Also disclosed herein are methods for manufacturing microarrays. In someembodiments, the microarrays disclosed herein can be synthesized in situon a surface, e.g., the substrate disclosed herein. In some instances,the microarrays are made using photolithography. For example, thesubstrate is contacted with a photoactive coupling solution. Masks canbe used to control radiation or light exposure to specific locations ona surface provided with free linker molecules or free coupling moleculeshaving protecting groups. In the exposed locations, the protectinggroups are removed, resulting in one or more newly exposed reactivemoieties on the coupling molecule or linker molecule. The desired linkeror coupling molecule is then coupled to the unprotected attachedmolecules, e.g., at the carboxylic acid group. The process can berepeated to synthesize a large number of features in specific orpositionally-defined locations on a surface (see, for example, U.S. Pat.No. 5,143,854 to Pirrung et al., U.S. Patent Application PublicationNos. 2007/0154946 (filed on Dec. 29, 2005), 2007/0122841 (filed on Nov.30, 2005), 2007/0122842 (filed on Mar. 30, 2006), 2008/0108149 (filed onOct. 23, 2006), and 2010/0093554 (filed on Jun. 2, 2008), each of whichis herein incorporated by reference).

In some embodiments, a method of producing a three-dimensionalmicroarray of features, can include obtaining a porous layer attached toa surface; and attaching the features to the porous layer, said featureseach comprising a collection of peptide chains of determinable sequenceand intended length, wherein within an individual feature, the fractionof peptide chains within said collection having the intended length ischaracterized by an average coupling efficiency for each coupling stepof at least about 98%. In some embodiments, the features are attached tothe surface using a photoactive coupling formulation, comprising aphotoactive compound, a coupling molecule, a coupling reagent, apolymer, and a solvent. In some embodiments, the features are attachedto the surface using a photoactive coupling formulation disclosedherein. In some embodiments, the photoactive coupling formulation isstripped away using water.

In one embodiment, described herein is a process of manufacturing anmicroarray. A surface comprising attached carboxylic acid groups isprovided. The surface is contacted with a photoactive coupling solutioncomprising a photoactive compound, a coupling molecule, a couplingreagent, a polymer, and a solvent. The surface is exposed to ultravioletlight in a deep ultra violet scanner tool according to a pattern definedby a photomask, wherein the locations exposed to ultraviolet lightundergo photo base generation due to the presence of a photobasegenerator in the photoactive coupling solution. The expose energy can befrom 1 mJ/cm² to 100 mJ/cm² in order to produce enough photobase.

The surface is post baked upon exposure in a post exposure bake module.Post exposure bake acts as a chemical amplification step. The bakingstep amplifies the initially generated photobase and also enhances therate of diffusion to the substrate. The post bake temperature can varybetween 75° Celsius to 115° Celsius, depending on the thickness of theporous surface, for at least 60 seconds and not usually exceeding 120seconds. The free carboxylic acid group is coupled to the deprotectedamine group of a free peptide or polypeptide, resulting in coupling ofthe free peptide or polypeptide to the carboxylic acid group attached tothe surface. This surface may be a porous surface. The synthesis ofpeptides coupled to a carboxylic acid group attached to the surfaceoccurs in an N→C synthesis orientation, with the amine group of freepeptides attaching to carboxylic acid groups bound to the surface of thesubstrate. Alternatively, a diamine linker may be attached to a freecarboxylic acid group to orient synthesis in a C→N direction, with thecarboxylic acid group of free peptides attaching to amine groups boundto the surface of the substrate.

The photoactive coupling solution can now be stripped away. In someembodiments, provided herein is a method of stripping the photoresistcompletely with deionized (DI) water. This process is accomplished in adeveloper module. The wafer is spun on a vacuum chuck for, e.g., 60seconds to 90 seconds and deionized water is dispensed through a nozzlefor about 30 seconds.

The photoactive coupling formulation may be applied to the surface in acoupling spin module. A coupling spin module can typically have 20nozzles or more to feed the photoactive coupling formulation. Thesenozzles can be made to dispense the photoactive coupling formulation bymeans of pressurizing the cylinders that hold these solutions or by apump that dispenses the required amount. In some embodiments, the pumpis employed to dispense 5-8 cc of the photoactive coupling formulationonto the substrate. The substrate is spun on a vacuum chuck for 15-30seconds and the photoactive coupling formulation is dispensed. The spinspeed can be set to 2000 rpm to 2500 rpm.

Optionally, a cap film solution coat is applied on the surface toprevent the non-reacted amino groups on the substrate from reacting withthe next coupling molecule. The cap film coat solution can be preparedas follows: a solvent, a polymer, and a coupling molecule. The solventthat can be used can be an organic solvent like N-methyl pyrrolidone,dimethyl formamide, or combinations thereof. The capping molecule istypically acetic anhydride and the polymer can be polyvinyl pyrrolidone,polyvinyl alcohol, polymethyl methacrylate,poly-(methyl-isopropenyl)-ketone, or poly-(2-methyl-pentene-1-sulfone).In some embodiments, the capping molecule is ethanolamine.

This process is done in a capping spin module. A capping spin module caninclude one nozzle that can be made to dispense the cap film coatsolution onto the substrate. This solution can be dispensed throughpressurizing the cylinder that stores the cap film coat solution orthrough a pump that precisely dispenses the required amount. In someembodiments, a pump is used to dispense around 5-8 cc of the cap coatsolution onto the substrate. The substrate is spun on a vacuum chuck for15-30 seconds and the coupling formulation is dispensed. The spin speedcan be set to 2000 to 2500 rpm.

The substrates with the capping solution are baked in a cap bake module.A capping bake module is a hot plate set up specifically to receivewafers just after the capping film coat is applied. In some embodiments,provided herein is a method of baking the spin coated capping coatsolution in a hot plate to accelerate the capping reactionsignificantly. Hot plate baking generally reduces the capping time foramino acids to less than two minutes.

The byproducts of the capping reaction are stripped in a strippermodule. A stripper module can include several nozzles, typically up to10, set up to dispense organic solvents such as acetone, isopropylalcohol, N-methyl pyrrolidone, dimethyl formamide, DI water, etc. Insome embodiments, the nozzles can be designated for acetone followed byisopropyl alcohol to be dispensed onto the spinning wafer. The spinspeed is set to be 2000 to 2500 rpm for around 20 seconds.

This entire cycle can be repeated as desired with different couplingmolecules each time to obtain a desired sequence.

In some embodiments, a microarray comprising a surface of freecarboxylic acids is used to synthesize polypeptides in an N→Corientation. In one embodiment, the carboxylic acids on the surface ofthe substrate are activated (e.g., converted to a carbonyl) to allowthem to bind to free amine groups on an amino acid. In one embodiment,activation of carboxylic acids on the group of the surface can be doneby addition of a solution comprising a carbodiimide or succinimide tothe surface of the microarray. In some embodiments, carboxylic acids canbe activated by addition of a solution comprising1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC),N-hydroxysuccinimide (NHS), 1,3-diisopropyl-carbodiimide (DIC),hydroxybenzotriazole (HOBt),O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU),benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), or N,N-diisopropylethylamine (DIEA) to the surface of themicroarray. The activation solution is washed away and the surface ofthe microarray is prepared for addition of an amino acid layer (i.e.,one amino acid at each activated carboxylic acid group). Carboxylic acidgroups remain activated for up to 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.

Addition of a solution comprising an amino acid with a free amine groupto the activated carboxylic acid surface of the microarray results inbinding of a single amino acid to each carboxylic acid group. In someembodiments, the amino acid comprises an amino acid with protected aminegroups. Using a photosensitive chemical reaction, the protecting groupcan be removed from the amine group of selected amino acids atsite-specific locations using a reticle. For example, Fmoc-protectedamino acids are mixed in a solution comprising a photobase. Uponexposure of the solution on the microarray to a specific frequency oflight at site-specific locations, the photobase will release a basewhich will deprotect the amino acid, resulting in coupling of the aminoacid to the activated carboxylic acid group on the surface of themicroarray. Another method of generating a base is through the use of aphotoacid generator. In some embodiments, the photoacid generator isN-boc-piperidine or 1-boc-4-piperazine.

After a completed layer of amino acids is coupled, remaining uncoupledactivated carboxylic acids are capped to prevent nonspecific binding ofamino acids on subsequent synthesis steps. The steps of activation,addition of an amino acid layer, and capping are repeated as necessaryto synthesize the desired polypeptides at specific locations on themicroarray.

In one embodiment, peptides synthesized in the N→C terminus directioncan be capped with a diamine molecule to enhance binding properties ofselected polypeptide sequences to a biological molecule, e.g., anantibody. In other embodiments, peptides synthesized in the C→Ndirection can be capped with a dicarboxylic acid molecule to enhancebinding properties of selected sequences to a biological molecule.

While synthesizing polypeptides in parallel on the surface of amicroarray, the method described herein ensures complete activation ofcarboxylic acid on the surface of the microarray. Due to stability ofthe activated ester for an extended period of time, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore coupling cycles may be completed after a single activation step(e.g., to couple an entire layer of 2-25 or more different amino acidsat different locations on the microarray). As the coupling occurs duringhard bake (heating in a hot plate at 85-90° Celsius for 90 secondsimmediately after coating) and due to the presence of excess amino acidin the solution, complete 100% deprotection of Fmoc-protected amino acidmay not be required for significantly high coupling yields. Afteraddition of all amino acids and capping, all free activated carboxylicacids are either coupled or capped, thus resulting in high efficiencyand accuracy of polypeptide synthesis.

Methods of Use of Microarrays

Also disclosed herein are methods of using substrates, formulations,and/or microarrays. Uses of the microarrays disclosed herein can includeresearch applications, therapeutic purposes, medical diagnostics, and/orstratifying one or more patients.

Any of the microarrays described herein can be used as a research toolor in a research application. In one embodiment, microarrays can be usedfor high throughput screening assays. For example, enzyme substrates(i.e., peptides on a peptide microarray described herein) can be testedby subjecting the microarray to an enzyme and identifying the presenceor absence of enzyme substrate(s) on the microarray, e.g., by detectingat least one change among the features of the microarray.

Microarrays can also be used in screening assays for ligand binding, todetermine substrate specificity, or for the identification of peptidesthat inhibit or activate proteins. Labeling techniques, protease assays,as well as binding assays useful for carrying out these methodologiesare generally well-known to one of skill in the art.

In some embodiments, a microarray can be used to represent a knownprotein sequence as a sequence of overlapping peptides. For example, theamino acid sequence of a known protein is divided into overlappingsequence segments of any length and of any suitable overlapping frame,and peptides corresponding to the respective sequence segments arein-situ synthesized as disclosed herein. The individual peptide segmentsso synthesized can be arranged starting from the amino terminus of theknown protein.

In some embodiments, a microarray is used in a method wherein theantigenic representation of the microarray includes at least one regionwhere the whole antigen sequence of a known protein is spanned viaepitope sliding; the immunoactive regions of the antigen are determinedby contacting one or more clinical samples on the array or a pluralityof different microarrays, and the set of peptide sequences required torepresent the known protein antigen are reduced.

In some embodiments, a sample is applied to a microarray having aplurality of random peptides. The random peptides can be screened andBLASTed to determine homologous domains with, e.g., a 90% or moreidentity to a given antigenic sequence. In some embodiment, the wholeantigenic sequence can then be synthesized and used to identifypotential markers and/or causes of a disease of interest.

In some embodiments, a microarray is used for high throughput screeningof one or more genetic factors. Proteins associated with a gene can be apotential antigen and antibodies against these proteins can be used toestimate the relation between gene and a disease.

In another example, a microarray can be used to identify one or morebiomarkers. Biomarkers can be used for the diagnosis, prognosis,treatment, and management of diseases. Biomarkers may be expressed, orabsent, or at a different level in an individual, depending on thedisease condition, stage of the disease, and response to diseasetreatment. Biomarkers can be, e.g., DNA, RNA, proteins (e.g., enzymessuch as kinases), sugars, salts, fats, lipids, or ions.

Microarrays can also be used for therapeutic purposes, e.g., identifyingone or more bioactive agents. A method for identifying a bioactive agentcan comprise applying a plurality of test compounds to a microarray andidentifying at least one test compound as a bioactive agent. The testcompounds can be small molecules, aptamers, oligonucleotides, chemicals,natural extracts, peptides, proteins, fragment of antibodies, antibodylike molecules or antibodies. The bioactive agent can be a therapeuticagent or modifier of therapeutic targets. Therapeutic targets caninclude phosphatases, proteases, ligases, signal transduction molecules,transcription factors, protein transporters, protein sorters, cellsurface receptors, secreted factors, and cytoskeleton proteins.

In another embodiment, a microarray can be used to identify drugcandidates for therapeutic use. For example, when one or more epitopesfor specific antibodies are determined by an assay (e.g., a bindingassay such as an ELISA), the epitopes can be used to develop a drug(e.g., a monoclonal neutralizing antibody) to target antibodies indisease.

In one embodiment, also provided are microarrays for use in medicaldiagnostics. An array can be used to determine a response toadministration of drugs or vaccines. For example, an individual'sresponse to a vaccine can be determined by detecting the antibody levelof the individual by using a microarray with peptides representingepitopes recognized by the antibodies produced by the induced immuneresponse. Another diagnostic use is to test an individual for thepresence of biomarkers, wherein samples are taken from a subject and thesample is tested for the presence of one or more biomarkers.

Microarrays can also be used to stratify patient populations based uponthe presence or absence of a biomarker that indicates the likelihood asubject will respond to a therapeutic treatment. The microarrays can beused to identify known biomarkers to determine the appropriate treatmentgroup. For example, a sample from a subject with a condition can beapplied to a microarray. Binding to the microarray may indicate thepresence of a biomarker for a condition. Previous studies may indicatethat the biomarker is associated with a positive outcome following atreatment, whereas absence of the biomarker is associated with anegative or neutral outcome following a treatment. Because the patienthas the biomarker, a health care professional may stratify the patientinto a group that receives the treatment.

In some embodiments, a method of detecting the presence or absence of amolecule of interest (e.g., a protein, an antibody, or any other ligand)in a sample can include obtaining a microarray disclosed herein andcontacted with a sample suspected of comprising the molecule ofinterest; and determining whether the molecule of interest is present inthe sample by detecting the presence or absence of binding to one ormore features of the microarray.

In some embodiments, a molecule of interest can be detected within asample that has a volume that is less than or equal to 100, 50, 10, 5,1.5, or 1 μL. In some embodiments, the elapsed time from the samplecontacting to detection of a molecule of interest is less than 40, 30,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1minutes. In some embodiment, a molecule of interest can be detected at aconcentration in the contacted sample that falls within the range ofabout 1 pg/ml to 1,000 μg/ml.

In some embodiments, the protein of interest may be obtained from abodily fluid, such as amniotic fluid, aqueous humour, vitreous humour,bile, blood serum, breast milk, cerebrospinal fluid, cerumen, chyle,endolymph, perilymph, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus, peritoneal fluid, pleural fluid, pus, saliva,sebum, semen, sweat, synovial fluid, tears, vaginal secretion, vomit, orurine.

In some embodiments, a method of identifying a vaccine candidate caninclude obtaining a microarray disclosed herein contacted with a samplederived from a subject previously administered the vaccine candidate,wherein the sample comprises a plurality of antibodies; and determiningthe binding specificity of the plurality of antibodies to one or morefeatures of the microarray. In some embodiments, the features comprise aplurality of distinct, nested, overlapping peptide chains comprisingsubsequences derived from a source protein having a known sequence.

Remote Microarray Analysis

In some embodiments, a diagnostic device comprising a chip array islocated in a third party location (e.g., a reference lab or a diagnosticlab). In some embodiments, the assay is performed in one or several ofthe third party locations and the patient samples are barcoded. The rawdata output from the chip array is input into an analyzer at auser-controlled location. This transfer may be through a VPN or via anyother remote data transfer method. In some embodiments, the raw data isstored in a temporary user-controlled database for a finite time. A testreport is generated and the results are provided to the third party. Anyadditional information requested by the third party may also be providedfrom additional analysis of the stored raw data. A flow diagramdepicting one embodiment of the diagnostic model provided herein isshown in FIG. 6.

In some embodiments, the analysis provides information on, for example,disease presence, disease severity, subtype of disease, the presenceand/or identity of multiple diseases and/or predisposition to diseases.In some embodiments, the analysis provides information from multiplexingdifferent antibody tests, multiple analytes from the same disease,multiplexing tests for different diseases. In some embodiments, theassay is an antibody-antigen interaction assay, a peptide-peptideinteraction assay, a peptide-protein interaction assay, aprotein-protein interaction assay, or a kinase interaction assay. Insome embodiments, the assay station is a fully or semi-automated roboticliquid handling station.

In some embodiments, after the test is complete, the raw data with nobar-code that never can be retraced is stored in a user-controlleddatabase (one way storage). This non-retraceable raw data will be usedto study the variability of the specific tests across populations andalso see the correlation between different antigenic peptide analyte indesigned-set to determine limits for the controls.

In some embodiments, a yearly subscription is provided to trend a set ofkey antigenic peptides representing different diseases to build aself-baseline for individual patients. In one embodiment of this method,the same person is tested on the same set of designed antigenic peptidesthat are biologically relevant or molecular mimicry, at different timeframe to trend whether or not a predisposition or early detection of adiseases to identify the trend change with solid evidence of key diseaserelated antigenic-peptides move from self-baseline and light up even toslightly higher level from the trend-range. Any improvements to thetrending subscription based designed-set will always have the legacy ofthe earlier designed-set so the continuity to trend of the same personis not lost, but improved with new addition to reflect progress indiagnostics.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current addition); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology(S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Multiple Linker Molecules for Protein, Peptide, or AntibodyAttachment to a Single Chip

This example describes the structure of selected linker molecules forattachment to a chip. The linker molecules will attach (“link”) aprotein, peptide, or antibody to the chip. The structure of the linkermolecule will affect the attachment of the protein, peptide, or antibodyto the chip and affect the binding affinity of the protein or antibodyto other proteins, peptides, or antibodies.

Silicon wafers were obtained from University wafers. A thin film ofNickel about 1000 Ångstrom thick was deposited on the wafers usingplasma-enhanced chemical vapor deposition (PECVD). This was followed byPECVD deposition of 500 Ångstrom thick layer of nitride. The nitride wassilinated using aminopropyl triethoxysilane (APTES) (FIG. 7, Linker 1).This silination step was immediately followed by coupling of aboc-protected Gly-PEG linker chain (FIG. 7, Linkers 2-8), where Glyrepresents a Glycine linker chain, and PEG represents polyethyleneglycol. The coupling was performed using simple Merrifield chemistry.Multiple other linker molecules can be attached onto a microarray tobind other peptides or proteins. Eight examples of linker configurationsare shown in FIG. 7. Linker 1 is silane-(boc), where (boc) represents atert-butyloxycarbonyl protecting group. Linker 2 is silane-Gly-PEG(boc).Linker 3 is silane-Gly-PEG-PEG(boc). Linker 4 is silane-Gly-(PEG(boc))₂.Linker 5 is silane-PEG-Gly(boc). Linker 6 is silane-Gly-cyc-PEG(boc),where Gly-cyc represents a glycine chain with a cyclic glycine chainconformation. Linker 7 is silane-Gly-(PEG(boc))₄. Linker 8 is cyclicpeptide loop formed by side chains of multiple Lysine and Glutamic acidmolecules. The use of multiple different linker molecules for the sameprotein allows one to determine the affinity and avidity of thebiological interactions. Linker molecules are not limited to thisexample, but can include, e.g. multiple lysine branches that are used toattach a protein or peptide to the silicon wafer chip. FIG. 8 shows thecontrol acetylated (CAP) versions of the linker molecules from FIG. 7.FIG. 9 shows the unprotected control linker molecules of FIG. 7 withoutthe tert-butyloxycarbonyl protecting group.

Example 2 Coupling of Anti-p53 Antibody and IL-6 Protein to a Chip

A wafers was first coated with the desired linker molecules from Example1 and a solution of 10% by weight polyvinylpyrrolidone (PVP) in water(FIG. 10, Step 1), followed by covering with a 5% Polyethylene glycol(PEG) based photoresist (FIG. 10, Step 2) and subsequent exposure withlight from a Nikon NSR 203 at 50 mJ/cm² (FIG. 10, Step 3). Afterexposure the wafers were developed with water (FIG. 10, Step 4). Theexposed areas were cross-linked while the non-exposed areas weredeveloped with water. The features of multiple different linkers wereused for coupling the IL-6 protein (FIG. 10, Step 5), wherein IL-6protein coupling solution was prepared as follows: 0.05 mg/ml of IL-6protein is dissolved in water along with 1 mg/ml of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 10% by weightof PVP. This coupling solution was then spin coated at 1000 rpm onto thewafer to obtain a uniform coat. The wafer was then baked at 37° Celsiusfor 5 minutes in a hot plate (FIG. 10, Step 6). Alternatively, thecoupling solution was dispensed and the wafer is stored at 4° Celsiusovernight to complete the protein coupling to the linker molecules.Next, the wafer was washed with water to strip the polymeric coat (FIG.10, Step 8). This completed the coupling of the first protein to thewafer. Since there are multiple types of linker molecules, the IL-6protein was available at varying concentration to determine the affinityand avidity of biological binding. The binding of the protein to linkermolecules attached to the surface via EDC coupling is shown in moredetail in FIG. 11.

Now the wafer was coated with 10% by weight PVP in water (FIG. 10, Step9) followed by 5% by weight PEG-based photoresist (FIG. 10, Step 10)before exposure with light from a Nikon NSR 203 at 50 mJ/cm² (FIG. 10,Step 11). After exposure the wafer was developed with water (FIG. 10,Step 12). The exposed areas were cross-linked while the non-exposedareas were developed with water. Now the wafer was spin coated with ap53 antibody coupling solution (FIG. 10, Step 13). p53 antibody couplingsolution consisted of 0.05 mg/ml of p53 antibody in water along with 1mg/ml of EDC and 10% by weight of PVP. This coupling solution was spincoated at 1000 rpm to obtain a uniform coat. The wafer is then baked at37° Celsius for 5 minutes on a hot plate (FIG. 10, Step 14).Alternatively, the p53 antibody coupling solution was dispensed and thewafer is stored at 4° Celsius overnight to complete the antibodycoupling to the linker molecules. Next, the wafer was washed with waterto strip the polymeric coat (FIG. 10, Step 16). This completed thecoupling of p53 antibodies. Since there are multiple types of linkermolecules, the same p53 antibody was available at varying concentrationto determine the affinity and avidity of biological binding. The bindingof the antibody to linker molecules attached to the surface via EDCcoupling is shown in more detail in FIG. 12.

Using the method described above, multiple different proteins can becoupled to a wafer in a high throughput method. The wafers were thendiced into small chips of a size of 1 mm by 1 mm. The diced chips wereplaced into the wells of a 364-well plate.

Example 3 Chip Array with Well Plates Using PDMS Film

A glass slide, 1 inch by 3 inch in size, of borosilicate was obtainedfrom VWR International. The thickness of the glass slide was 1.2 mm. APDMS film with a minimum depth of 5 mm and a maximum depth of 9 mm wasattached to the glass slide after plasma treatment of the glass slide.The thickness of the well walls was 1 mm. In this example, the size ofthe chip was 20 mm by 20 mm with the wells sizes measuring 4 mm by 4 mm.The edges were flat in this example. In an alternative example, theedges were tapered so that the film can easily be stripped off from theglass slide by manual or machine peeling. Subsequently, epoxy glue,EPO-TEK 301 from Epoxy Technology, was applied to the glass slide insidethe wells where the chips were to be attached. With a pick-and-placerobot or manually, the chips were placed on top of the epoxy glue andcured at room temperature for 24 hours. The same procedure was appliedto the chips placed in the other wells. Now the glass slides were placedinto frames of a standard 384-well plate and snapped on tight. Theprocess for developing this chip array is shown in FIG. 13. A top viewof the array of wells for holding chips produced by the process above isshown in FIG. 14. A side view of the chip array of wells is shown inFIG. 15. To perform a bio assay, the chip arrays were fed into aHamilton Robotics liquid assay station. Once the wash and dry steps werecompleted in this assay station, the PDMS film was peeled off eithermanually or using a robot. The chips were then scanned to determine theresults of the assay.

Example 4 Chip Array with Well Plates Using Inverted Plate Pillars

A polypropylene standard 24-, 96- or 384-well plate is used. The platelid was a custom made injection molded using stainless steel to achievea flatness of around 10 μm on the base and on top of the plate pillars.The diagram in FIG. 16 shows an example of the required configuration.Part 1 is a typical well plate. Part 2 is an inverted lid with platepillars to hold the chips. The chips can either be attached to theseplate pillars using epoxy glue or using part 3. In both cases, the chipswere bonded tight to the substrate. In a typical liquid assay station,the wells were first filled with the required buffer solutions. Then apick-and-place robot handled the plate lid and immerses the chipsexactly into the desired wells. The height of the plate pillars wasaround 2-3 mm whereas the depth of the wells was around 5-6 mm so thatthe required amounts of buffer solution or samples when added did notspill over to the neighboring well. This amount was determined to be 100μl. After the entire bio assay procedure was completed, the plate lid isturned upside down so the chips face upwards and were then dried using apurge of nitrogen. The chips were then scanned in a confocal scannermicroscope. The stage of the confocal scanner microscope has thecapability to hold the frame of a standard well plate. Multiple chipscan be scanned using this set up.

Example 5 Chip Loading onto Caps and Cap Attachment to Plate Pillars

In this example, each wafer was coupled with only 1 protein using themethodology described in Example 2 above. For example, one 9 square inchwafer coupled with protein was diced into 1 mm×1 mm chips, resulting inapproximately 52,000 chips carrying the same protein, e.g. p53 protein.The wafer can also be diced into chips of sizes ranging from 0.5 mm² to10 mm². The protein chips were assembled onto protein chip caps, asshown in FIG. 17A. A plate with plate pillars as shown in FIG. 17Binterfaces with the caps, allowing each cap to snap onto each platepillar. The top of the caps consist of a plurality of chip holders witheach chip. The minimum spacing between the chip holders ranges fromapproximately 0.25 mm to approximately 5 mm. A chip was mounted on a capusing a high throughput automated surface mount technology (SMT)pick-and-place machine. The plate pillars with caps mounted are shown inFIG. 17C. The maximum of components per hour (cph) that can be mountedvia the SMT pick-and-place machine ranged from about 20,000 cph to about150,000 cph. The resulting caps with mounted multiple protein chiparrays can be used for example with a 24-pillar or 96-pillar plate for abinding affinity assay (i.e., a serum assay). Example dimensions of a24-pillar and 96-pillar plate are shown in FIGS. 17D and 17E,respectively.

Example 6 Assay Probing Anti-p53 Antibody and IL-6 Protein on Chip Array

The pillar plates holding protein chip arrays (as described above) wereused in a serum assay to determine antibody and protein binding. For theassay sample serum is added to a chip with immobilized p53 antibody andIL-6 according to Example 2 following the general steps of: First, thechips were washed in Phosphate Buffered Saline with Tween-20 (PBST)thrice while gentle shaking the solution. Then, the chips were incubatedwith serum from different patients in a 384-well plate at 37° Celsius.Subsequently, the chips were washed thrice with PBST before incubatingthe chips with secondary antibodies to detect the targetantibody-antigen binding. The chips were again washed thrice with PBSTbefore scanning the chips in a Nikon AIR confocal or CCD scannermicroscope.

The scanner microscope included a CCD camera with an image chip that hasa 2048×2048 pixel resolution and frame size of 1 mm×1 mm. The frame rateof the CCD camera can be adjusted within the range of 15-30frames/second, and typically set at 30 frames/second. To complete thedata acquisition during a scan 16 frames were line integrated to yieldone shot of the scanned area. The fluorescence probes attached to thesecondary antibodies were each excited at wavelengths of 488 nm and 470nm with the emission wavelengths of 525 nm and 670 nm, respectively.This allowed for the detection of two different antibodies each carryinga different fluorescence probe. To eliminate for example detection atthe wavelength of 670 nm a band-pass filter in the wavelength range of550±5 nm can be employed to filter out the emission at 670 nm. In orderto distinguish among different features on a chip a resolution of 4×4pixels per feature is used. The results of the assay are shown in FIGS.18-22.

FIG. 18 shows the results of an assay using a chip comprising IL-6immobilized to the surface of the chip with different linker molecules,as described in Example 2. Recombinant IL-6 and polyclonal antibodieswith a high binding affinity for IL-6 were obtained from LifeTechnologies. The variation in binding affinity of anti-IL-6 antibody toIL-6 was tested across multiple types of linkers on a single chip. IL-6was coupled to the chip as explained in Example 2. Polyclonal rabbitantibody to human IL-6 was added to the chip at a dilution of 1:1000 inPBS. The secondary antibody which binds to the IL-6 antibody was goatanti-rabbit alexafluor 488. The chip was incubated in the dark for 1hour to allow for the antibody to bind. FIG. 18 confirms IL-6 proteinbinding to multiple linker molecules on the chip. The linking sites withthe highest binding affinity are ones with 4 PEGs attached to Gly.

FIG. 19 shows the results from an assay with a serum comprisinganti-IL-6 antibodies performed on protein array chips with immobilizedp53 antibody and IL-6 as described above. The binding regions showedsimilar results as seen in FIG. 18. The acetylated control linkersshowed none or minimal binding and the linker molecules with the largestnumber of NH₂ groups displayed the highest binding affinity.

FIG. 20 shows the results of an assay using a chip comprising anti-p53antibodies. Anti-p53 polyclonal mouse antibody and recombinant human p53protein were obtained from ABCAM. The variation in binding of anti-p53antibody to p53 protein is tested across multiple linker moleculesattached to a single chip. Anti-p53 antibody was coupled to the chip asdescribed in Example 2. Polyclonal mouse antibody to human p53 proteinwas added at a dilution of 1:1000 in PBS. The secondary antibody is goatanti-mouse alexafluor 488. The chip was incubated for 1 hour in the darkto allow for the antibody binding. The results shown in FIG. 20confirmed that anti-p53 antibody binds to multiple linker moleculesattached to the chip. The highest binding affinity of anti-p53 antibodywas observed at sites that include linker molecules with 4 PEGs attachedto Gly.

FIG. 21 shows results from the assay with p53 protein serum performed onprotein array chips with immobilized p53 antibody and IL-6 as describeabove. The binding regions showed similar results to those observed inFIG. 20. The acetylated control linkers showed none or minimal bindingand the linker with regions having the largest number of NH₂ groupsshowed the highest binding affinity.

FIG. 22 shows only minimal binding to control acetylated linkermolecules when no protein or antibody are attached to any of the linkermolecules attached to the chip.

A diagram of the assay illustrating the various binding events is shownin FIG. 23. When the serum was applied to the chip, both the protein andthe antibody bound to the corresponding molecules coupled to the chip.Secondary antibodies comprising a fluorophore were used to detect thebinding of the protein or antibody to the antibody or protein originallyimmobilized onto the chip surface via linker molecules.

Example 7 Assay Probing IL-6 Protein on 96-Pillar Plate

In this example, the assay protocol includes 96 chips, each sized 10mm×10 mm and mounted on a separate pillar of a 96-pillar plate. For theassay TBS buffer, PBST buffer and BSA were obtained from VWRInternational and antibodies were obtained from ABCAM. Six 96-wellplates were prepared beforehand with well plate no. 1 containingmethanol, well plate no. 2 containing TBS buffer, well plate no. 3containing primary antibody, well plate no. 4 containing PBST buffer,well plate no. 5 containing secondary antibody and well plate no. 6containing DI water.

A 96-pillar plate containing the chips was consecutively placed in wellplate no. 1 for 5 minutes, in well plate no. 2 for 5 minutes, in wellplate no. 3 and incubated at 37° Celsius for 30 minutes, in plate no. 4for 5 minutes, in well plate no. 5 and incubated at 37° Celsius for 30minutes, in well plate no. 5 for 5 minutes and in well plate no. 6 for 5minutes for a total assay time of 85 minutes.

The benefits of this assay process include: The overall assay time wasreduced by avoiding the need for removing the previous solution andadding the next solution for each assay step, since all well plates wereprepared before the assay is performed. Furthermore, the volume ofanti-IL-6 antibody required for completely covering one chip array onthe pillar plate was 10 μl compared to 25 μl required for a conventionalassay using regular microarrays being placed in well plates. Finally,the number of peptides or features immobilized on each chip isapproximately 2,000,000 which allows for more data to be collected froma single chip and better data analysis. Table 1 compares the assayinvolving chips on a 96-pillar plate with a conventional microarrayassay.

TABLE 1 Assay Comparison Assays Parameters 96-pillar plate conventionalassay Time for assay completion 85 minutes 160 minutes Antibody quantity10 μl/chip 25 μl/well Number of tested peptides 2,000,000/chip135,000/well

A more detailed comparison of the performance of various pillar plateconfigurations is presented in Table 2.

TABLE 2 Assay Performance Pillar Plate Comparison Chip No. of detectedScan and Features/ No. of Chip size per features Assay analysissample/sec pillars (mm²) pillar (min-max) throughput throughput(min-max) 24 100 1 2m-18m 20 mins/4 plates 2.8 hr/4 plates 19047-17142896 20.25 1 400k-3.5m  20 mins/4 plates 2.8 hr/4 plates 15238-133333 3844 1 81k-720k 20 mins/4 plates 2.8 hr/4 plates 12342-109714 1536 1.56 130k-270k 20 mins/4 plates 2.8 hr/4 plates 18292-164634 24 0.16 400120k-1m   20 mins/4 plates 2.8 hr/4 plates 1142-9523  96 0.16 8125k-225k 20 mins/4 plates 2.8 hr/4 plates 952-8571 384 0.16 16 5k-45k 20mins/4 plates 2.8 hr/4 plates 761-6857 1536 0.16 4 1k-9k  20 mins/4plates 2.8 hr/4 plates 609-5487 m = millions; k = thousands.

Example 8 Variations and Performance in Assays Probing p53 TAD1 onPillar Pates

This Example describes a variation of the above assay protocol inExample 6 includes using anti-p53 antibody and growing the p⁵³ 10aaTAD1transcription activation factor having the amino acid sequenceLKWLDSFTEQ on the chips of pillar plates. The sequence is listed in thereverse order, since the peptide is synthesized on each chip in multiplelocations starting from the N-terminus. The assay determined variationin results obtained from multiple locations within one chip(intra-chip), from the same location between two chips (inter-chip),from chip locations within chips that were mounted on pillars located atthe same position among different plates (intra-pillar), and from chiplocations within chips on pillars located at different position on aplate (inter-pillar). To ensure that these locations coincide among thedifferent chips during the measurement, the chips were aligned based onalignment marks as described in more detail below.

Intra-chip variations in intensities were measured by analyzing the dataobtained for different locations on one chip by detecting the p53 TAD1peptide using the assay protocol as described above. The intra-chipvariations in intensities listed in Table 3 were obtained from a singlechip.

TABLE 3 Intra-chip intensity variation Location Binding Intensity 161245 2 62030 3 61075 4 61145 5 61324

Inter-chip variations in intensities were measured by analyzing the dataobtained from five chips with the data collected at five differentlocations for each chip. Again the results indicated the binding ofanti-p53 antibody to p53TAD1 peptide immobilized at locations 1-5 oneach chip according to the assay protocol as described above. Theinter-chip variations in intensities listed in Table 4 were obtainedfrom a single pillar plate.

TABLE 4 Inter-chip intensity variation Chip1 Chip2 Chip3 Chip4 Chip5Location Intensity Intensity Intensity Intensity Intensity 1 61075 6098760471 60001 61245 2 60985 60147 61025 61234 62030 3 61040 60023 6100361147 61075 4 61075 60754 61002 61074 61145 5 61115 61712 60965 6046561324

Intra-pillar variations in intensities were measured by analyzing thedata obtained from five chips on five different plates with the datacollected from five different locations on each chip. In addition, thefive pillars holding the chips were located at the same position withina 96-pillar plate when comparing the plates with each other. Theintra-pillar variations in intensities listed in Table 5 were obtainedfrom a five chips on five different 96-pillar plates.

TABLE 5 Intra-pillar intensity variation Chip1 Chip2 Chip3 Chip4 Chip5Location Intensity Intensity Intensity Intensity Intensity 1 60570 6198561748 60223 60040 2 60985 62005 61025 60368 61000 3 61010 60425 6100261085 60000 4 61005 60586 61056 61096 60963 5 60999 60789 60458 6032560332

Inter-pillar variations in intensities were measured by analyzing thedata obtained from five chips on five different pillars at differentpositions on the same 96-pillar plate. In addition the data wascollected from five different locations on each chip. The inter-pillarvariations in intensities listed in Table 6 were thus obtained from afive chips on a single 96-pillar plate.

TABLE 6 Inter-pillar intensity variation Chip1 Chip2 Chip3 Chip4 Chip5Location Intensity Intensity Intensity Intensity Intensity 1 60765 6045661039 61042 61355 2 60452 62010 60564 62014 61421 3 60125 61078 6102660642 61014 4 60352 60987 62356 60933 60784 5 60332 61020 60789 6000060332

The intensity data from Tables 3-6 was then averaged over the fivelocations and chips for each experiment. The experimental average(=mean) in intensity variations including the mean±variation % arelisted in Table 7, where the variation % was calculated by dividing thestandard deviation by the mean of the collected data.

TABLE 7 Intensity variations averaged over locations and chips VariationType Intensity (Mean ± Variation %) Intra-chip variation 61363.8 ± 0.56%Inter-chip variation 60964.76 ± 0.69%  Intra-pillar variation  60843.2 ±0.857% Inter-pillar variation 60915.52 ± 0.938%

Example 9 Sensitivity of Chip to Antibody Concentration

In this example, the sensitivity of a single 10 mm by 10 mm sized chipwas measured with respect to increases in antibody concentration. Theassay protocol is similar to the one described in Example 8 usinganti-p53 antibody upon coupling a p53 10aaTAD1 peptide to the chipsurface. The confocal scanner microscope can measure a relativeintensity of up to 65,536 (=2¹⁶) when using an image chip that containeda resolution of 16-bits per pixel. With a maximum measurable intensityof 65,536 the scanner was capable of measuring a change in concentrationcovering 4 Log orders. Increasing the resolution to 20 bits per pixelyielded a maximum signal resolution of 1,048,576 (=2¹⁰) with aconcentration sensitivity of 6 Log orders (FIG. 24). Measurements of theintensity changes by increasing the antibody concentration are listed inTable 8, including the intensity signal of a single feature, theintensity averaged among all features on the chip and the correspondingvariation % that was calculated by dividing the standard deviation bythe mean (=average) of the collected feature intensity data.

TABLE 8 Contentration dependency of intensity (chip sensitivity)Concentration Intensity Average Intensity Variation % 1 ng/ml 3,0243,146 1.14% 100 ng/ml 18,202 19,117 1.04% 500 ng/ml 31,456 32,438 1.64%1 μg/ml 35,564 36,765 1.42% 2 μg/ml 46,132 48,453 1.43% 5 μg/ml 66,69269,327 1.46% 10 μg/ml 85,293 88,794 1.07% 50 μg/ml 150,900 154,631 1.52%100 μg/ml 193,012 200,694 1.09% 500 μg/ml 342,400 356,100 1.87% 750μg/ml 395,429 415,289 1.38% 1,000 μg/ml 430,131 455,861 1.90%

To confirm reproducibility, the same experiment was twice repeated fortwo different chips (Chip 1 and Chip 2). With an R² of 0.9997 and 0.9992among the two experiment for each chip, respectively, and an R² of0.9982 among the two chips the sensitivity of each chip was repeatedlyreproduced (FIG. 25), and thus is reliable to a high degree.

Example 10 Analysis of Confocal or CCD Scanner Microscope Signal OutputScanning an Ultra High Feature Density Chip Array

This example describes the analysis of a chip array by identifyingregions of interest for piecewise scanning, correcting scanned framesfor misalignment among chips, and stitching the data of each frame toaccount for frame overlaps. A 24-pillar or 96-pillar plate was placed onthe stage of a Nikon AIR confocal microscope or CCD scanner microscope.The 24-pillar and 96-pillar plates were as described in Example 5 andcontained multiple chips per chip array mounted on each plate pillar.The chips were pre-aligned during mounting on the chip array with apre-alignment accuracy of about ±50 μm as shown in the actual chiplayout in FIG. 26. To get the data from the scanned frames withoutmissing any feature rows or columns, data stitching is important. Fordata stitching to be accurate, the alignment of the scanned framesrequired a high degree of accuracy. A higher accuracy was achieved byrotating the stage around its centre (coinciding with the plate centre)before scanning a chip to correct for any misalignment of the chipwithin respect to other chips on the plate.

Identification of ROIs on Microarray

Refraction differences due to the composition of surface of a chipcontaining substrate pillars substrate (as described above) was used toaccurately determine regions of interest (ROIs) for the analysis of achip array. The contrast between the metal layer and the substrate(silica/nitride) where the probe molecules (features) were presentprovided for intensity to be picked up using reflected light from aconfocal laser source. This enabled piece-wise scanning of differentmicroarrays on a chip array. The image from the reflected light was usedto map out different ROIs on a chip. One or more laser sources alongwith a scanner was used to excite emission and capture the reflected andemitted light for picking up signal intensities from differentfluorophores located within various ROIs. An image of the reflectedlight and images of the red and green emission light channels from asingle chip are shown in FIG. 27.

The reflected light image from each frame map out where the fluorophoreintensities need to be measured. By setting a threshold for thereflected light image one can distinguish ROIs containing features frombackground areas on a chip as shown in FIG. 28. Background areas containmetal that generated a different contrast compared to the pillarsubstrate so that one easily differentiated those areas from the ROIareas of features. The data was collected for each frame only from ROIareas (defined by the reflected light map and containing features) whichcan be characterized by the stage coordinate X and stage coordinate Yposition for each feature along with its averaged fluorophore intensitymeasured at one or multiple wavelengths (multiplex) at the same time.

A flowchart illustrating the process of determining an intensitythreshold for identifying ROIs on a chip is shown in FIG. 29. Theso-determined ROIs on the chip are shown in FIG. 30.

Correction for Chip Misalignment

In order to process multiple chips on a chip array, alignment marks wereused to correct for any misalignment among the chips caused duringpre-alignment. Each chip was marked with squared alignment marks thatwere at least 1.5 times larger than any squared feature area on a chipas shown in FIG. 31. The chip boundary was defined by four alignmentmarks. Thus, a software program can recognize alignment marks andfeature areas based on size. Coordinates of alignment marks were used asplate stage coordinates, since the gaps between chips are much smallerthan chip size with more than one chip being scanned at a time. Forexample shown in FIG. 32, 9 alignment marks (a-g) fall within thescanned frame if the chip was not centered within the frame and areaoccupied by the chip and its surrounding gaps was smaller than the framearea. To distinguish between alignment marks from the same and otherchips, the distance between any scanned alignment marks was determinedand only alignment marks (a-c) with distances being approximately equalto chip size belong to the same chip. For some chip, all four alignmentmarks (e-f, g, and h-i) did not fall within the same frame area. Theprocess then searched for the remaining alignment marks in adjacentframes.

In another example, with the chip size far exceeding the frame size eachchip had two alignment marks on two edges as shown in FIG. 33. Eachalignment mark contained 5 points P1, P2, P3, P4 and P5 as a crosspattern, wherein the line connecting P1 to P2 and the line connecting P3and P4 shared middle cross point P5. Instead of using a cross patternthese five points were located using an algorithm that differentiatedthe pattern from the background using a intensity histogram or the sizeof the alignment mark pattern as discussed above. The CCD camera thendetected the five points (P1, P2, P3, P4, and P5) and a software programcompared these points with the standard reference positions (P1′, P2′,P3′, P4′, and P5′) with P5′ being in the frame center.

In both examples, a software program then calculated a rotation angletheta (θ) and offset translation to correct for the misalignment ofalignment mark with respect to a standard reference position of the chiparray as illustrated in FIGS. 34 and 35. The relationships between thetaand various distance parameters were given by:

${{\tan \; \theta} = {\frac{AO\_ Y}{AO\_ X} = \frac{FO\_ Y}{FO\_ X}}},{{\cos \; \theta} = {\frac{AO\_ X}{AD} = \frac{FO\_ X}{FD}}},{{\sin \; \theta} = {\frac{AO\_ Y}{AD} = \frac{FO\_ Y}{FD}}},{{AD} = \sqrt{{AO\_ X}^{2} + {AO\_ Y}^{2}}},{{FD} = \sqrt{{FO\_ X}^{2} + {FO\_ Y}^{2}}},$

with AD being the alignment distance, AO_X the alignment offset alongthe X-axis, AO_Y the alignment offset along the Y-axis, FD the featuredistance, FO_X the feature offset along the X-axis, and FO_Y the featureoffset along the Y-axis. The subsequent steps for correcting formisalignment among chips on a chip array are shown in FIGS. 36-39 basedon aligning the pillar plate by rotating the stage around its centre.

Frame Stitching

Data from each frame was then stitched together with each adjacent framewith two adjacent frames overlapping by 5-10% or 0.1 mm as shown in theexample in FIG. 30. To determine the positions of all features on achip, based on the alignment coordinates the corner feature of a chipwas first located as shown in FIG. 40. From the corner feature, thelocation of next feature was calculated using FO_X and FO_Y. Subsequentfeatures were located by repeatedly moving along the X- and Y-axis insteps of FO_X and FO_Y.

The features having the same global coordinates were then data-stitchedin real time rather than image-stitched which eliminates time anddiscrepancies due to stitching images. Real time stitching included thatafter an image was acquired, the data was pulled out and stored in anexternal file or data storage that was continuously and simultaneouslyappended (stitched) when another image was scanned. The total time forscanning and stitching images of an ultra high density microarray withmore than 500 k features took less than 10 minutes as compared to 35minutes using image-processing software, e.g. Genepix. A high throughputmultithreading algorithm further reduced this total time of scanning andstitching to the range of a few seconds.

FIG. 41 illustrates a flow chart summarizes the various steps of dataanalysis of an ultra high feature density chip array.

Example 11 Quality Control Monitoring System

This example describes inline and end-of-line quality control (QC)monitoring systems that assure high quality chips with ultra highfeature density.

Inline Quality Control

The benefit of an inline quality control monitoring system that correctmeasures can be taken before the end of the manufacturing line isreached was demonstrated in this Example. This increased the throughputefficiency and decreased the manufacturing time by maintain a high yieldof high quality chips.

Inline Quality Control of Chip Array Thickness

In FIG. 42, the wafer was coated in step 1 with a photobase solutioncontaining photobase, polymer and amino acid and soft baked at 85°Celsius for 90 seconds. In the next control step 2 (QC1), the waferthickness was measured and monitored for comparison with the expectedspecifications (Table 9). If the thickness measured was not within thestandards specified, further processing of the wafer was stopped and thewafer was stripped, recoated and followed by repeating steps 1 and 2.The wafer was then exposed using a photomask at 248 nm in step 3 beforebeing hard backed at 85° Celsius for 90 seconds in step 4. In the laststep 5 (QC2), the wafer thickness was again measured and monitored forcomparison with the expected specifications (Table 9). If the thicknessmeasured was not within the standards specified, further processing ofthe wafer was stopped with the wafer being stripped and recoated andthen repeating steps 1 through 5 again.

TABLE 9 Pass Criteria for Thickness QC Step Thickness threshold QC1 2400nm ± 30 nm QC2  800 nm ± 10 nm

In this example, the inline QC monitoring system was tested on a set of15 wafers while continually monitoring the thickness for each wafer. Theresults of the test are listed in Table 10 with only two wafers failingthe QC1 and additional two failing the QC2 step.

TABLE 10 Inline QC Test Results Wafer # QC1 QC2 Status W1 2402 nm 795 nmPassed W2 2415 nm 792 nm Passed W3 2385 nm 805 nm Passed W4 2444 nm —Failed QC1 W5 2398 nm 810 nm Passed W6 2385 nm 795 nm Passed W7 2407 nm802 nm Passed W8 2425 nm 815 nm Failed QC2 W9 2430 nm 794 nm Passed W102415 nm 804 nm Passed W11 2375 nm 797 nm Passed W12 2450 nm — Failed QC1W13 2403 nm 808 nm Passed W14 2400 nm 799 nm Passed W15 2398 nm 820 nmFailed QC2

Inline Diffusion and Overlay Quality Control

An additional inline QC monitoring step included checking for diffusionand overlay variations. In this example, after the wafer was exposedusing a photomask at 248 nm and baked, the diffusion pattern of thephotobase is regulated using a standard microscope to determine if thepattern matches the expected standard pattern as follows: For thismonitoring step a standard test diffusion and overlay pattern was etchedinto photomask at predetermined locations as shown in FIG. 43A.

The distance between each dot was 100 nm and was equidistant in +X, −X,+Y and −Y direction. If the wafer was exposed under standard conditions,the pattern under the microscope appeared as shown in FIG. 43B (standardpattern after exposure and bake). In case of any derivation fromstandard conditions as a result from a difference in exposure energy orplacement of the wafer, the results deviated from the standard diffusionand overlay pattern, respectively. Some test cases for correct andincorrect overlay and diffusion amount are listed in Table 11 andillustrated in FIGS. 44A-C, wherein an incorrect overlay or diffusionpattern would deviate by more than 5% from the standard pattern,respectively. Only the correct diffusion and overlay pattern shown inFIG. 44A was accepted and the wafer continued processing. In any othercase, the processing of the wafer was stopped with the wafer beingstripped, recoated and reassessed with the inline QC monitoring system.

TABLE 11 Diffusion and overlay test results FIG. 44A Correct diffusion &overlay pattern FIG. 44B Incorrect overlay variation in +X directionFIG. 44C Incorrect overlay variation in +Y direction FIG. 44D Incorrectdiffusion amount variation

End-of-Line Quality Control

After the completion of processing the wafer in the inline QC monitoringsystem, end-of-line quality control was performed to further assure highquality of the chip. In this example three end-of-line quality controltests were described with the first one assessing the couplingefficiency, i.e. peptide synthesis, and the last two testing thebiological performance of the synthesized peptide.

Quality Control Test 1

For each processing step, there were at least 2 (up to 25) differentfeatures which were also exposed using the photomask during the inlineQC monitoring system. The amino acid coupling for each feature wasmeasured separately using fluorescein coupled to each feature todetermine the coupling efficiency. The results were then compared with athreshold pass criteria for each feature listed in Table 12 to determinethe status of each wafer.

TABLE 12 Pass criteria for amino acid coupling Amino Acid NameFluoroscein Intensity Threshold AA1 64125 ± 1000 AA2 63500 ± 1000 AA363000 ± 1000 AA4 64520 ± 1000 AA5 64185 ± 1000

In this example, the results of testing a set of five processed wafersare listed in Table 13 with only one wafer failing in both chiplocations. If the wafer passed the threshold intensity criteria for allpre-determined locations, then it was further processed in the nextend-of-line quality control step.

TABLE 13 Test results for amino acid coupling Wafer # AA1 AA2 AA3 AA4AA5 Status Location 1 W1 64100 63500 63125 65505 65025 Passed W2 6401564000 62985 65520 63456 Passed W3 64155 63220 62330 64985 63789 PassedW4 64550 63363 63890 64275 64010 Passed W5 62250 64055 63998 63998 64185Failed AA1 Location 2 W1 64120 63505 63128 65525 65028 Passed W2 6301564087 62980 65563 63456 Passed W3 64175 63202 62315 64941 63777 PassedW4 64520 63363 63847 64212 64080 Passed W5 61650 64095 63900 63902 64112Failed AA1

Quality Control Test 2

The next end-of-line quality control step comprised testing thebiological performance of particular peptides coupled to the wafer usinga set of commercially available monoclonal antibodies. A set of at least5 (up to 75) different monoclonal antibodies were tested by synthesizingtheir corresponding antigen peptide sequences on the wafer. Thebiological performance of the wafer was then tested and compared withthe threshold criteria for antibody binding listed in Table 14.

TABLE 14 Pass criteria for antibody binding Antibody Name Intensity CA164050 ± 1200 CA2 55789 ± 1000 CA3 64125 ± 1000 CA4 60150 ± 1000 CA562335 ± 1000

In this example, the results of testing a set of five processed wafersare listed in Table 15 with only one wafer failing. If the wafer passedboth end-of-line quality control test (Test 1 and 2), it was consideredof high quality and ready for use.

TABLE 15 Test results for antibody binding Wafer # CA1 CA2 CA3 CA4 CA5Status W1 64020 56880 64532 60985 63015 Passed W2 63255 55020 6478959963 61789 Passed W3 63985 54996 65020 60125 62785 Passed W4 6378556125 63889 61002 62145 Passed W5 64785 50252 64025 60178 62330 FailedCA2

Quality Control Test 3

An additional end-of-line quality control step comprised testing thebiological performance of peptides coupled to the wafer by usingcommercially available antibodies. First, for each commercial antibodyit was determined whether a particular amino acid in a peptide sequenceis material for binding the antibody. An amino acid was consideredmaterial when the biological activity of the corresponding peptide ishigh if the amino acid is part of the peptide as compared to low whenthe peptide lacks the amino acid that is replaced by any other aminoacid. Various antibody peptide sequences were grown on a chip todetermine at least one peptide sequence in which each of the amino acidswas material to that peptide for binding a particular antibody.

For example, considering the sequence LKWLDSFTEQ, L was replaced one ata time by an amino acid selected from the group consisting of CIT, A, C,D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W and Y. Subsequently, theremaining amino acids in the sequence were similarly replaced one at atime. All sequences were then synthesized on the wafer and were testedfor biological activity using the commercial antibody for the abovesequence.

In this example, goat anti-rabbit IgG, goat anti-mouse IgG and alltested commercial antibodies, including anti-citrulline antibody (ab100932), were obtained from ABCAM. TBS Buffer, PBST Buffer and BSA wereobtained from VWR International. In the assay the chips containing allsequences were mounted on a 96-pillar plate and washed with methanol for5 minutes followed by washing with TBS Buffer for 5 minutes. Primaryantibody solution containing PBST and 1% BSA was incubated on the chipat 37° Celsius for 1 hour. The chip was then washed with PBST for 5minutes thrice, followed by secondary antibody incubation at 37° Celsiusfor 1 hour, wherein the secondary antibody solution contained PBST, 1%BSA, and the goat anti-rabbit IgG or goat anti-mouse IgG depending onthe primary antibody being used. The chip was washed with PBST for 5minutes thrice, followed by washing with DI water for 5 minutes twice.

FIGS. 45A-K illustrates assay results for the following sequences:LKWLDSFTEQ, DKYYEPHLERA, AATNSSTIAGHK, AYDPVDYPY, SSQPLPLARENK,LNLVEELPK, PSLSLAFKTQFD, EAPKAEAGDAKG, NKVGSYAVSNNA, EVTDEFMEKSAM,DGYDFETTTDYD, respectively (bolded and italicized amino acids werematerial for antibody binding). For example, from the intensity mapshown in FIG. 45A, KWLDS were the material amino acids for antibodybinding in the LKWLDSFTEQ sequence. If any other amino acid was used inplace of these amino acids, the sequence did not show any biologicalactivity for antibody binding.

This experiment correlated an amino acid with a particular sequence forwhich it is a material amino acid. When this particular amino acid wasthen grown in a layer of the wafer during peptide synthesis, thecorrelated sequence was also grown as a test sequence in the design tocheck the coupling yield by evaluating the biological activity of thetest sequence.

For example, K was a material amino acid for the sequence LKWLDSFTEQ.Thus, whenever K was grown for each layer during the peptide synthesis,there was a corresponding test sequence LKWLDSFTEQ in the design. If 12layers of K were grown, there were 12 different locations on the waferat which the corresponding test sequence was grown. FIG. 46 shows theresults of binding intensity for the different K polymers employing anassay as described above and varying the anti-p53 antibodyconcentration.

In another example, biological performance for the amino acid citrulline(CIT) was validated using a sequence containing citrulline (YAT6SSP) andanti-citrulline antibody that reacted specifically with a peptidecontaining citrulline irrespective of any other amino acid presentsequence. The chips contained both sequences with one that containedcitrulline (YAT6SSP) and the other that lacked citrulline (YATRSSP) andacted as a mutant sequence. This sequence was grown for each layer forwhich citrulline was added during the peptide synthesis and biologicalperformance of citrulline was tested for each synthesis step (meraddition) using the anti-citrulline antibody.

In summary, an end-of-line QC monitoring system validated wafers usingfluorescein coupling and evaluated biological performance of each aminoacid added during the synthesis of a peptide chain on the wafer.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. A method for obtaining feature binding data, comprising: obtaining amicroarray, said microarray comprising at least 100,000 features persquare centimeter; contacting said microarray with a sample comprising aplurality of ligands for at least a subset of said 100,000 features persquare centimeter under conditions that promote ligand binding; andimaging said microarray to identify binding of said plurality of ligandsto said features of said microarray.
 2. The method of claim 1, wherein atotal number of features is at least about 500,000, at least about1,000,000, at least about 2,000,000, or at least about 18,000,000. 3.-5.(canceled)
 6. The method of claim 1, wherein said microarray has an areathat is less than or equal to 0.2 square millimeters, less than or equalto 1 square millimeter, less than or equal to 10 square millimeters,less than or equal to 100 square millimeters, or less than or equal to150 square millimeters. 7.-10. (canceled)
 11. The method of claim 1,wherein said sample has a volume that is less than or equal to 100 μL,is less than or equal to 50 μL, is less than or equal to 25 μL, is lessthan or equal to 10 μL, is less than or equal to 5 μL, is less than orequal to 1.5 μL, or is less than or equal to 1 μL. 12.-17. (canceled)18. The method of claim 1, wherein an elapsed time from samplecontacting to finishing the imaging is less than 20 minutes, less than 5minutes, or is equal to or less than 1 minute. 19.-20. (canceled) 21.The method of claim 1, wherein an elapsed time for imaging is equal orless than 20 seconds, equal or less than 10 seconds, or equal or lessthan 1 second. 22.-23. (canceled)
 24. The method of claim 1, wherein acoefficient of variation of data obtained from said microarray is notgreater than 5 percent, not greater than 2 percent, or not greater than1 percent. 25.-26. (canceled)
 27. The method of claim 1, wherein saidmicroarray comprises at least 1,000,000 features per square centimeter,at least 10,000,000 features per square centimeter, or at least15,000,000 features per square centimeter. 28.-29. (canceled)
 30. Themethod of claim 1, said contacting occurs at a concentration of saidplurality of ligands that is less or equal than 1,000 μg/ml in saidsample, less or equal than 10 μg/ml in said sample, less or equal than 1μg/ml in said sample, less or equal than 0.1 μg/ml in said sample, lessor equal than 0.1 μg/ml in said sample, less or equal than 10 ng/ml insaid sample, less or equal than 1 ng/ml in said sample, or less or equalthan 5 pg/ml in said sample. 31.-36. (canceled)
 37. The method of claim1, said contacting occurs at a concentration of said plurality ofligands that is within the range of approximately 1 pg/ml toapproximately 1,000 g/ml in said sample.
 38. The method of claim 1,wherein said imaging comprises identifying binding of at least 1,000ligands, at least 100,000 ligands, at least 1,000,000 ligands, at least10,000,000 ligands, at least 15,000,000 ligands, or at least 100,000,000ligands to said features of said microarray. 39.-43. (canceled)
 44. Themethod of claim 1, wherein said features are selected from a groupconsisting of proteins, DNA binding sequences, antibodies, peptides,oligonucleotides, nucleic acids, peptide nucleic acids, deoxyribonucleicacids, ribonucleic acids, peptide mimetics, nucleotide mimetics,chelates, and biomarkers.
 45. An inverted pillar plate for assayingmicroarrays, comprising: a plurality of chip mounts, each chip mountconfigured to affix at least one of a plurality of microarrays and toprevent the at least one microarray from being displaced from the chipmount when the chip mount is placed facing downwards into a wellcontaining an assay solution; and a plate comprising a plurality ofinverted plate pillars that extend approximately perpendicular from theplate, each inverted plate pillar configured to be coupled to one of theplurality of chip mounts, wherein each chip mount is affixed to at leastone of the plurality of inverted plate pillars so that each chip mountis prevented from being displaced from the at least one inverted platepillar when the plate is turned upside down. 46.-70. (canceled)
 71. Amethod for collecting data from chip arrays and for piecewise real-timescanning and stitching of said data, comprising steps of: providing achip array comprising of: a plurality of microarrays, each microarraycomprising features that are attached to a surface of the microarray atpositionally-defined locations, aligning a first region of the chiparray with a scan mask of a microscope; imaging the first region of thechip array under the scan mask by the microscope; and rotating, by usinga computer processor, the imaged first region of the chip array intostandard orientation based on an alignment mark on a surface of amicroarray that is at a positionally-defined location within the imagedfirst region.
 72. The method of claim 71 further comprising steps of:aligning a second region of the chip array with the scan mask so thatthe second region partially overlaps with the first region; imaging thesecond region of the chip array under the scan mask by the microscope;and rotating, by using a computer processor, the imaged second region ofthe chip array into standard orientation based on an alignment mark on asurface of a microarray that is at a positionally-defined locationwithin the imaged second region.
 73. The method of claim 72 furthercomprising steps of: combining the rotated images of the first andsecond region for analyzing the features located on the surface of themicroarrays within the imaged first and second region, wherein anyoverlapping parts of the imaged first and second region are averaged forthe analysis.
 74. A method of claim 72 further comprising steps of:storing the rotated images of the first and second region within animage database.